Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling

Julian M Carosi; Leanne K Hein; Jarrod J Sandow; Linh V P Dang; Kathryn Hattersley; Donna Denton; Sharad Kumar; Timothy J Sargeant

doi:10.1080/15548627.2023.2281126

. 2023 Nov 8;20(4):863–882. doi: 10.1080/15548627.2023.2281126

Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling

Julian M Carosi ^a,^b,^c, Leanne K Hein ^a, Jarrod J Sandow ^d,^e,^f, Linh V P Dang ^a, Kathryn Hattersley ^a, Donna Denton ^b, Sharad Kumar ^b,^g, Timothy J Sargeant ^a,^✉

PMCID: PMC11062367 PMID: 37938196

ABSTRACT

Retromer prevents the destruction of numerous receptors by recycling them from endosomes to the trans-Golgi network or plasma membrane. This enables retromer to fine-tune the activity of many signaling pathways in parallel. However, the mechanism(s) by which retromer function adapts to environmental fluctuations such as nutrient withdrawal and how this affects the fate of its cargoes remains incompletely understood. Here, we reveal that macroautophagy/autophagy inhibition by MTORC1 controls the abundance of retromer⁺ endosomes under nutrient-replete conditions. Autophagy activation by chemical inhibition of MTOR or nutrient withdrawal does not affect retromer assembly or its interaction with the RAB7 GAP protein TBC1D5, but rather targets these endosomes for bulk destruction following their capture by phagophores. This process appears to be distinct from amphisome formation. TBC1D5 and its ability to bind to retromer, but not its C-terminal LC3-interacting region (LIR) or nutrient-regulated dephosphorylation, is critical for retromer to be captured by autophagosomes following MTOR inhibition. Consequently, endosomal recycling of its cargoes to the plasma membrane and trans-Golgi network is impaired, leading to their lysosomal turnover. These findings demonstrate a mechanistic link connecting nutrient abundance to receptor homeostasis.

Abbreviations: AMPK, 5’-AMP-activated protein kinase; APP, amyloid beta precursor protein; ATG, autophagy related; BafA, bafilomycin A₁; CQ, chloroquine; DMEM, Dulbecco’s minimum essential medium; DPBS, Dulbecco’s phosphate-buffered saline; EBSS, Earle’s balanced salt solution; FBS, fetal bovine serum; GAP, GTPase-activating protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; LANDO, LC3-associated endocytosis; LP, leupeptin and pepstatin; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; nutrient stress, withdrawal of amino acids and serum; PDZ, DLG4/PSD95, DLG1, and TJP1/zo-1; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; SLC2A1/GLUT1, solute carrier family 2 member 1; SORL1, sortillin related receptor 1; SORT1, sortillin 1; SNX, sorting nexin; TBC1D5, TBC1 domain family member 5; ULK1, unc-51 like autophagy activating kinase 1; WASH, WASH complex subunit.

KEYWORDS: Autophagy, MTOR, MTORC1, retromer, TBC1D5, VPS35

Introduction

Retromer is an evolutionarily conserved assembly of VPS35 (VPS35 retromer complex component), VPS26 (paralog A or B), and VPS29 that orchestrates the endosomal sorting and trafficking of numerous receptors to the trans-Golgi network or plasma membrane [1–6]. By doing so, retromer limits the lysosomal turnover of these receptors and is critical for their homeostasis and signaling functions [5,6]. Retromer is incorporated into distinct sub-complexes that associate with SNX1 (sorting nexin 1)-SNX2 and SNX5-SNX6, SNX3, and SNX27 [7–11]. The endosomal recruitment of retromer is regulated by RAB7A^GTP [12–14]. On the endosomal surface, retromer recognizes sorting motifs on the cytoplasmic tail of its cargo receptors including those with the sequence Trp-Leu-Met as well as DLG4/PSD95, DLG1, and TJP1/zo-1 (PDZ) motifs [11,15,16]. Following the spatial concentration of receptor cargoes, retromer engages with the WASH complex to “push” these endosomal domains outward through local F-actin polymerization prior to scission [17]. To facilitate receptor recycling, these carriers are targeted to the trans-Golgi network by SNX-BAR- or SNX3-retromer, or to the plasma membrane by SNX27-retromer [7,10,17–19]. Following this, retromer is thought to be uncoated from endosomes by its interactor TBC1D5 (TBC1 domain family member 5), which is a GTPase activating protein (GAP) for RAB7A and RAB7B [13,20,21].

The endosomal network converges with other degradative pathways within the cell such as autophagy at the level of the amphisome or lysosome [22]. Autophagy captures intracellular materials (e.g., damaged organelles, protein aggregates, and invading pathogens) and delivers them to the lysosome for destruction and recycling of their constituent macromolecules, a process that is necessary for an organism to overcome starvation [22]. Autophagy is primarily inhibited by the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) and is stimulated by 5’-AMP-activated protein kinase (AMPK) which together elicit control over the autophagy initiation machinery including ULK1 (unc-51 like autophagy activating kinase 1) [23]. MTORC1 and AMPK are regulated by nutritional and energetic cues, respectively, and this enables autophagy to remobilize nutrient stores by lysosomal catabolism when either is lacking [23–26]. Retromer also regulates autophagy in several key ways: by (1) promoting MTORC1-dependent autophagy inhibition [19,27]; (2) trafficking of autophagy machinery such as ATG9A (autophagy related 9A) [28,29]; (3) supporting autophagosome-lysosome fusion [30,31]; and (4) ensuring the degradative capacity of lysosomes [19,30,32,33].

The selection of substrates during autophagy is carried out by several autophagy receptors including SQSTM1/p62, TAX1BP1, and NBR1 [34]. These receptors physically link substrates to lipidated mammalian Atg8-family proteins (such as MAP1LC3/LC3A, LC3B, and LC3C, and GABARAP, GABARAPL1 and GABARAPL2; referred to here as ATG8 proteins) that incorporate into the expanding phagophore via an ATG8/LC3-interacting region (LIR) ([W/F/Y]xx[L/I/V], where x denotes any amino acid) [34]. The retromer regulator TBC1D5 contains several putative LIRs, however, it remains unclear whether it acts as an autophagy receptor and, if so, for what cargoes? The C-terminal LIR (₈₀₉FTIV₈₁₂) of TBC1D5 is critical for binding to ATG8 proteins in vitro and for targeting it to autophagic vesicles in cells [35,36]. Its N-terminal LIR (₅₉WEEL₆₂) is required for binding to the retromer component VPS29 and this interaction can be outcompeted by increasing amounts of LC3A in vitro [35,36]. Autophagy-dependent uncoupling of TBC1D5 from retromer during glucose deprivation enhances the interaction between TBC1D5 and ATG8 proteins and its recruitment to autophagic vesicles [37]. This switch is believed to relieve the inhibitory effect of TBC1D5 over retromer to promote recycling of the retromer cargo SLC2A1/GLUT1 (solute carrier family 2 member 1) to the plasma membrane to maximize glucose uptake [37,38]. Similarly, autophagy activation by catalytic inhibition of MTOR, but not partial allosteric inhibition of MTORC1 with rapamycin, weakens the TBC1D5-retromer interaction and increases the interaction between TBC1D5 and ATG8 proteins as well as other autophagy effectors including ULK1, ATG9A, and ATG13 [37–39]. This has led to the hypothesis that TBC1D5 acts as a molecular switch to amplify cargo sorting and trafficking during autophagy [35,37–39]. Despite this, a mechanistic explanation of how retromer function and receptor homeostasis is coordinated by autophagy remains incomplete.

Here, we reveal a mechanism that inhibits receptor recycling by coordinating TBC1D5-dependent capture of retromer⁺ endosomes by autophagosomes for wholesale destruction. This nutrient-sensitive and MTORC1-regulated process appears to be distinct from amphisome formation, and provides insight into how nutrient availability coordinates receptor homeostasis.

Results

Autophagy inhibition by MTORC1 controls the abundance of retromer⁺ endosomes

To investigate how the endosomal network adapts to nutrient fluctuations we examined how endosomes decorated with the receptor recycling complex retromer respond to nutrient stress (withdrawal of amino acids and serum). In HeLa cells, nutrient stress with Earle’s balanced salt solution (EBSS) led to marked reduction of VPS35⁺ vesicles throughout the cytoplasm (Figure 1A,B). Similar changes were observed in nutrient-replete conditions following exposure to the MTOR inhibitors AZD8055 (catalytic inhibitor of MTORC1 and MTORC2) and rapamycin (allosteric inhibitor of MTORC1) (Figure 1C,D). As expected, phosphorylation of RPS6 (ribosomal protein S6) S240/244 by RPS6KB1/S6K1 downstream of MTORC1 was suppressed by nutrient stress, AZD8055, and rapamycin (Figure 1E) [40–43]. While each appeared to induce autophagy, as shown by an increased ratio of lipidated to free LC3B (LC3B-II:LC3B-I), the extent of induction with rapamycin was weakest, likely because inhibitory phosphorylation of ULK1 S758 by MTORC1 was insensitive to rapamycin (Figure 1E), as previously reported [41]. Phosphorylation of ULK1 S555 by AMPK was markedly increased by nutrient stress but not chemical inhibition of MTOR (Figure 1E) [44]. Phosphorylation of AKT S473 by MTORC2 was inhibited by AZD8055 but not nutrient stress or rapamycin, consistent with short-term rapamycin exposure failing to inhibit MTORC2 assembly (Figure 1E) [45,46]. Thus, MTORC1 inhibition was enough to activate autophagy and to remodel VPS35⁺ endosomes. Despite changes in the number of retromer⁺ endosomes, nutrient stress and MTOR inhibition did not affect the total amount of VPS35 protein in cell lysates (Figure 1E). Glucose deprivation moderately reduced the phosphorylation of RPS6KB1 T389 by MTORC1 (Figure S1A) [47]; it also reduced the number of VPS35⁺ vesicles, and when combined with AZD8055 led to a further decrease in vesicle number (Figure S1B and S1C). Thus, nutrients (i.e., amino acids and serum, and glucose) control the abundance of retromer⁺ endosomes, most likely through an MTORC1-dependent mechanism given that rapamycin did not inhibit MTORC2 in the experimental timeframes tested (Figure 1E).

Figure 1. — Autophagy inhibition by MTORC1 controls the abundance and distribution of retromer⁺ endosomes. (A) nutrient stress reduces the abundance of retromer-decorated endosomes. Cells were treated with or without EBSS for 6 h prior to immunofluorescence with a VPS35 antibody. Blue, DAPI stained nuclei. Cell outlines are indicated. Scale bar: 10 µm. (B) VPS35⁺ vesicles per cell from (A). Values are the mean ± SEM from n = 10–11 fields/condition from three independent experiments (unpaired t-test). (C) MTORC1 inhibition reduces the abundance of retromer-decorated endosomes. Cells were treated with or without AZD8055 or rapamycin for 6 h prior to immunofluorescence with a VPS35 antibody. Blue, DAPI-stained nuclei. Cell outlines are indicated. Scale bar: 10 µm. (D) VPS35⁺ vesicles per cell from (C). Values are the mean ± SEM from n = 10–25 fields/condition from three independent experiments (one-way ANOVA with Tukey’s multiple comparisons test). (E) the impact of nutrient stress and pharmacological inhibitors on MTOR and AMPK signalling. Immunoblot of lysates from cells treated with or without EBSS, AZD8055 or rapamycin for 6 h. Blots were probed for the indicated proteins and phosphoproteins. (F) MTORC1 inhibition downstream of nutrient stress reduces the abundance of retromer-decorated endosomes. Cells transfected with RHEB^Y35N-FLAG or an empty vector were treated with or without EBSS for 6 h prior to immunofluorescence with VPS35 and FLAG antibodies. Blue, DAPI stained nuclei. Scale bar: 10 µm. (G) VPS35⁺ vesicles per cell from (F). Values are the mean ± SEM from n = 41–115 cells/condition from three independent experiments (two-way ANOVA with Tukey’s multiple comparisons test). (H) loss of ATG5 impairs lipidation of ATG8 proteins following autophagy activation. Immunoblot of lysates from wild-type and *ATG5*-null cells treated with or without AZD8055 or EBSS for 6 h. Blots were probed for the indicated proteins. (I) loss of ATG5 prevents nutrient stress and MTOR inhibition from regulating the abundance of retromer-decorated endosomes. Wild-type and *ATG5*-null cells were treated with or without AZD8055 or EBSS for 6 h prior to immunofluorescence with a VPS35 antibody. Blue, DAPI stained nuclei. Scale bar: 10 µm. (J) VPS35⁺ vesicles per cell from (I). Values are the mean ± SEM from n = 44–51 cells/condition from three independent experiments (two-way ANOVA with Tukey’s multiple comparisons test).

To explore this possibility, we overexpressed a FLAG-tagged constitutively active mutant of the MTORC1 activator RHEB^Y35N that renders the MTORC1 pathway resistant to inhibition by nutrient stress [48,49], as indicated by the phosphorylation of RPS6KB1 T389 by MTORC1 in HEK293T cells (Figure S1D), as well as RPS6 at S235/236 and S240/244 by RPS6KB1 in HeLa cells (Figure S1E and S1F) [50]. Importantly, in HeLa cells, RHEB^Y35N-FLAG expression prevented EBSS-dependent changes in the abundance of VPS35⁺ vesicles (Figure 1F,G). Thus, MTORC1 inhibition by nutrient stress is important for coordinating the abundance of retromer⁺ endosomes.

To determine whether autophagy was regulating retromer downstream of MTORC1 inhibition, ATG5 was deleted from HeLa cells via CRISPR-Cas9 gene-editing. ATG5 is part of an E3-like complex that catalyzes the lipidation of ATG8 proteins (such as LC3B) which is critical for proper construction and maturation of autophagosomes as well as for substrate selection during autophagy [51,52]. Loss of ATG5 prevented LC3B lipidation and this was unchanged following the activation of autophagy with either EBSS or AZD8055 (Figure 1H) or lysosomal inhibition with chloroquine (CQ) (Figure S1G). Under basal conditions, ATG5-null cells resembled wild-type cells exposed to AZD8055 or EBSS as the amount of VPS35⁺ vesicles were reduced (Figure 1I,J). However, unlike wild-type cells, loss of ATG5 prevented AZD8055- and EBSS-dependent reductions in VPS35⁺ vesicle number (Figure 1I,J). Because MTORC1 signaling is moderately reduced when autophagy is inactivated – via loss of ATG5 (Figure S1H-J) or lysosomal inhibition (Figure S1K and S1L) – disentangling autophagy inhibition from MTORC1 inhibition was challenging. While other explanations are possible (such as autophagy-independent functions), these findings in combination with those described later, led us to suspect that retromer may be coordinated by autophagy downstream of MTORC1 inhibition.

The retromer-TBC1D5 interaction is not nutrient sensitive

To explore how MTORC1 and/or autophagy elicit control over retromer, we examined how the VPS35 interactome is rewired by nutrient stress and pharmacological inhibition of MTOR. First, we confirmed that VPS35-FLAG expressed in VPS35-null cells responded to nutrient stress in a similar way as the endogenous protein (Figure S2A and S2B). Immunoprecipitation of VPS35-FLAG from cells treated with or without AZD8055 or EBSS enabled determination of VPS35 interactome rewiring by label-free proteomics (Figure 2A-D and Table S1). This approach revealed significant enrichment of retromer components (VPS35, VPS26A and -B, and VPS29), TBC1D5, and WASH complex components (WASH3P, WASH2P, WASHC1/WASH1, WASHC5/KIAA0196, WASHC4/KIAA1033, WASHC2A/FAM21A, and WASHC3/CCDC53) in untreated conditions compared with the background control (i.e., untreated VPS35-null cells) (Figure 2B). Other retromer interactors such as SNX27 and ANKRD27/VARP showed minor but non-significant enrichments (log₂ fold-change over background ~ 2.3 and ~ 1.2, respectively; P > 0.05) (Figure 2B). Importantly, these core members of the VPS35 interactome were largely resistant to re-wiring by nutrient stress and MTOR inhibition (Figure 2C,D), which was validated by immunoblot analysis of co-immunoprecipitated proteins (Figure 2E).

The RAB7 GAP TBC1D5 was the most abundant VPS35 interactor (log₂ fold-change over background ~ 10.8) (Figure 2B-D). Neither TBC1D5 nor VPS35 was necessary for maximal stability of the other (Figure 2F), although VPS35 but not TBC1D5 was necessary for the stability of VPS26 and VPS29 (Figure 2E,G). Consistent with TBC1D5 acting downstream of retromer recruitment to endosomes, its loss did not inhibit VPS35 recruitment to vesicles throughout the cytoplasm whereas loss of VPS35 prevented vesicle recruitment of TBC1D5 (Figure 2H) [13]. In VPS35-null cells, stable co-expression of VPS35-FLAG and HA-TBC1D5 enabled crude assessment of the retromer-TBC1D5 interaction from anti-HA and -FLAG immunoprecipitations performed in parallel (Figure 2I). We found that while a large fraction of the total VPS35 pool is bound to TBC1D5, only a small fraction of the total TBC1D5 pool is bound to VPS35/retromer (Figure 2I). Importantly, our finding that the VPS35-TBC1D5 interaction was not disrupted during autophagy activation by nutrient stress or MTOR inhibition was unexpected because glucose deprivation was previously reported to block the retromer-TBC1D5 interaction in an autophagy-dependent manner (Figure 2B-E) [37,38]. Consistent with our assessment of the retromer interactome, TBC1D5 and VPS35 overlapped on vesicles under basal conditions and following combined treatment with AZD8055 and bafilomycin A₁ (BafA) (to induce autophagy while preventing lysosomal turnover), as well as in the absence of ATG5 (Figure 2J). Therefore, we concluded that the MTORC1 pathway regulates retromer vesicle abundance without impacting assembly of the complex or its interactions with key regulatory proteins (such as TBC1D5).

MTORC1 prevents capture and turnover of retromer-TBC1D5 decorated endosomes by autophagosomes

Given that nutrient stress and MTOR inhibition reduced the abundance of retromer⁺ endosomes (Figure 1A-D, J and 1K), we suspected that these endosomes may be turned over by autophagy. To induce autophagy while preventing the turnover of lysosomal substrates, cells were co-treated with AZD8055 and BafA which markedly increased the overlap of VPS35 with LC3B⁺ vesicles in HEK293T cells (Figure S3A), SH-SY5Y neuroblastoma cells (Figure S3B), as well as wild-type but not ATG5-null HeLa cells (Figure 3A). Next, we set out to determine if endosome redistribution to LC3B⁺ vesicles was specific to retromer⁺ endosomes. To this end, we compared EEA1⁺ early endosomes with maturing endosomes decorated the retromer component VPS26, as well as their intermediate compartment (EEA1⁺ and VPS26⁺) (Figure S3C). In HeLa cells stably expressing FLAG-LC3B, VPS26⁺ endosomes redistributed to FLAG-LC3B⁺ vesicles to a greater extent than EEA1⁺ early endosomes did following co-treatment with AZD8055 and BafA (Figure 3B,C). Remarkably, EEA1⁺ endosomes that redistributed to FLAG-LC3B⁺ vesicles were also VPS26⁺ (Figure 3B,C). This indicated that cells exhibit a preference for targeting retromer⁺ endosomes to the autophagic compartment following MTOR inhibition (Figure 3B,C).

Figure 3. — MTORC1 prevents capture and turnover of retromer-TBC1D5 decorated endosomes by autophagosomes. (A) VPS35 colocalizes with LC3B following MTOR inhibition but not in the absence of ATG5. Wild-type and *ATG5*-null cells were treated with or without AZD8055 for 6 h. AZD8055-treated cells were supplemented with BafA for the final 3 h prior to immunofluorescence with VPS35 and LC3B antibodies. Blue, DAPI stained nuclei. Scale bar: 10 µm. Insets show 2.5× magnification. A representative of three independent experiments is shown. (B) compared to EEA1⁺ endosomes, VPS26⁺ endosomes are preferentially targeted to LC3B⁺ vesicles following MTOR inhibition. Cells stably expressing FLAG-mTurbo-LC3B were treated with or without AZD8055 for 6 h. AZD8055-treated cells were supplemented with BafA for the final 3 h prior to immunofluorescence with EEA1, VPS26 and FLAG antibodies. Scale bar: 10 µm. Insets show 5× magnification. A representative of three biological replicates is shown. (C) colocalization of EEA1, VPS26, and EEA1 and VPS26 double-positive vesicles with FLAG-mTurbo-LC3B from (B). Values are the mean ± SEM from n = 29–33 cells/condition from three biological replicates (two-way ANOVA with Tukey’s multiple comparisons test). (D) VPS35 is recruited to autophagosomes following MTOR inhibition. Cells stably expressing RFP-GFP-LC3B to distinguish autophagosomes (GFP⁺ and RFP⁺) from autolysosomes or amphisomes (GFP⁻ and RFP⁺) were treated with or without AZD8055 for 6 h prior to immunofluorescence with a VPS35 antibody. Blue, DAPI stained nuclei. Scale bar: 10 µm. Insets show 2.5× magnification. A representative of two independent experiments is shown. (E) autophagosomes (GFP⁺ and RFP⁺) and autolysosomes/amphisomes (GFP⁻ and RFP⁺) per cell from (D). Values are the mean ± SEM from n = 19–20 cells/condition from two independent experiments (two-way ANOVA with Tukey’s multiple comparisons test). (F) VPS35⁺ autophagosomes (GFP⁺/RFP⁺) per cell from (D). Values are the mean ± SEM from n = 19–20 cells/condition from two independent experiments (mann-whitney U test). (G) the retromer-TBC1D5 complex becomes membrane-protected following MTOR inhibition. Post-nuclear lysates from cells treated with or without a combination of AZD8055 and BafA for 18 h were digested with proteinase K in the presence of absence of Triton X-100 to lyse intracellular membranes or were left untreated. Immunoblots were probed for the indicated proteins to indicate whether they resided within or outside intracellular vesicles/membranes. TAX1BP1 and SQSTM1 served as membrane-protected control proteins, and RPS6KB1 served as a non-membrane protected control protein. A representative of three independent experiments is shown.

To assess which type of LC3B⁺ compartment retromer was preferentially associating with, we employed HeLa cells that stably expressed RFP-GFP-LC3B to distinguish autophagosomes (GFP⁺ and RFP⁺) from autolysosomes and amphisomes (GFP⁻ and RFP⁺) [48,53]. MTOR inhibition with AZD8055 markedly increased the formation of autophagosomes (GFP⁺ and RFP⁺) while moderately reducing the pool of autolysosomes and/or amphisomes (GFP⁻ and RFP⁺) (Figure 3D,E). Interestingly, we found the retromer component VPS35 to mostly colocalize with autophagosomes (GFP⁺ and RFP⁺) following MTOR inhibition (Figure 3D,F). In some cases, multiple VPS35⁺ puncta colocalized with larger LC3B⁺ compartments indicating that retromer-coated endosomes were potentially captured by autophagosomes rather than the retromer complex itself undergoing a platform switch from endosomes to autophagosomes (Figure 3D and S3A). To explore this further, we employed a protease protection assay to establish whether the retromer-TBC1D5 complex was being recruited to the inside or outside of autophagosomes (Figure 3G) [54]. Typically, proteins within the lumen of membrane-bound compartments (e.g., autophagosomes) from post-nuclear cell lysates are protected from degradation by a protease supplemented in vitro [54]. In contrast, cytoplasmic proteins and proteins on the outer surface of intracellular membranes are accessible to the protease for degradation [54]. Akin to bona-fide autophagy receptors TAX1BP1 and SQSTM1 that translocate to the inside of autophagosomes during autophagy [34], the retromer components VPS35 and VPS26, as well as TBC1D5, shifted from being proteinase K-sensitive to -resistant following co-treatment of cells with AZD8055 and BafA (Figure 3G). Unexpectedly, VPS29 was proteinase K-resistant even under basal conditions (Figure 3G). We suspect that VPS29 may be intrinsically protease-resistant, similar to other proteins such as GAPDH [55] or inaccessible to the protease when incorporated within retromer and retriever complexes [56]. Unlike autophagy cargo receptors, the retromer-TBC1D5 complex did not exhibit basal flux into the membrane-protected compartment following lysosome inhibition with BafA (Figure S3D). Instead, their delivery into the membrane-protected compartment was largely stimulated by AZD8055 when lysosomal degradation was inhibited (Figure S3D). Since only a small fraction of the total retromer pool was preserved in the membrane-protected compartment, we wondered whether this reflected its relative abundance on endosomes. Indeed, using a subcellular fractionation approach, we revealed that only a small fraction of the total retromer pool is associated with the endosomal membrane while the majority exists “free” in the cytoplasm (Figure S3E). A similar distribution was noted for numerous other proteins known to cycle between endo-lysosomal membranes and the cytoplasm (Figure S3E). This may explain why we were unable to detect changes in the levels of retromer components in whole cell lysates following nutrient stress or MTOR inhibition. Altogether, our findings indicate that following MTORC1 inhibition, retromer-TBC1D5-decorated endosomes are captured by autophagosomes to facilitate their lysosome-mediated destruction.

The TBC1D5 interaction is critical for retromer to be trafficked to autophagosomes following MTOR inhibition

We speculated that TBC1D5 may target retromer⁺ endosomes to expanding phagophores following autophagy activation. Indeed, loss of TBC1D5 inhibited the redistribution of VPS35 to LC3B⁺ compartments following MTOR inhibition (Figure 4A,B). Thus, we concluded that TBC1D5 controls an autophagy-regulated switch in retromer localization. While the retromer-TBC1D5 interaction itself was not regulated by MTORC1, we suspected that their mobilization to autophagosomes may be coordinated by phosphorylation. Using mass spectrometry, we detected three evolutionarily conserved serine residues within TBC1D5 (S560, S561, and S566) that were dephosphorylated – while bound to retromer – following exposure to both nutrient stress and MTOR inhibition (Figure S4A-C). These residues occupied a region of TBC1D5 that is predicted to be unstructured (Figure 4C). We next exploited site-directed mutagenesis of TBC1D5 to identify key determinants of retromer mobilization to autophagosomes. In cells lacking TBC1D5, expression of wild-type HA-tagged TBC1D5 restored delivery of VPS35 to LC3B⁺ compartments following MTOR inhibition (Figure 4D-F). Both phospho-mimetic TBC1D5^{S560D,S561D,S566D} and phospho-deficient TBC1D5^{S560A,S561A,S566A} mutants behaved akin to the wild-type protein, leading us to conclude that de-phosphorylation of TBC1D5 at these sites is not necessary for retromer to be targeted to autophagosomes (Figure 4D-F). The TBC1D5^L142E mutant that cannot bind to retromer exhibited enhanced colocalization between VPS35 and LC3B⁺ compartments under basal conditions, but its delivery was not stimulated by MTOR inhibition (Figure 4D-F and S4D) [20].

Next, we reasoned that TBC1D5 may target retromer⁺ endosomes to expanding phagophores via its LIRs (Figure 4C). The iLIR database indicated that TBC1D5 contains three distinct LIRs (LIR #1 [₅₉WEEL₆₂], LIR #2 [₇₃₇FILI₇₄₀], and LIR #3 [₈₀₉FTIV₈₁₂]) (Figure 4C) [57]. Its N-terminal LIR #1 lies within a predicted α-helix (Figure 4C) and is critical for ATG8 binding in vitro but is also necessary for binding to VPS29 [35]. Thus, it is difficult to interpret in vivo functions of its N-terminal LIR #1 from mutagenesis studies. The C-terminal LIR #3 of TBC1D5 is similarly important for binding to ATG8 proteins in vitro, yet only combined deletion of LIR #1 and LIR #3 completely abolishes their interaction [35]. These in vitro findings indicate that LIR #2 is unlikely to be of functional importance. Unexpectedly, mutation of LIR #3 within TBC1D5^F809A,V812A that binds to ATG8 proteins in vitro – yet still interacts with retromer – was dispensable for targeting VPS35 to LC3B⁺ compartments (Figure 4D-F) [35]. Surprisingly, endogenous ATG8 proteins LC3B and GABARAP did not co-immunoprecipiate with HA-TBC1D5 (Figure 4G), and in the reverse experiment HA-TBC1D5 did not co-immunoprecipitate with overexpressed GFP-LC3B regardless of autophagy induction (Figure 4H). Prolonged exposure of the immunoblot identified a nonspecific interaction between GFP and HA-TBC1D5 (Figure 4H). Similarly, while TBC1D5 was previously reported to interact with the autophagy activator ULK1 [39], we found ULK1 to be a major contaminant in our anti-HA immunoprecipitations (Figure 4G). In sum, we conclude that mobilization of retromer to autophagosomes in response to MTOR inhibition requires its ability to bind to TBC1D5, but not its C-terminal LIR #3 or dephosphorylation.

Retromer does not interact with RAB7A

The overall reduction in retromer⁺ vesicles during MTOR inhibition likely occurred via autophagy-mediated turnover but may be explained in part by reduced recruitment of retromer to endosomes. In a current model of retromer regulation, RAB7A^GTP binds to retromer and recruits it to endosomes to facilitate receptor sorting [13]. Following this, retromer recruits TBC1D5 to hydrolyze RAB7A^GTP via its GAP activity leading to the disassociation of retromer from endosomes [4,12,13]. While our prior epistasis experiments indicated that TBC1D5 acts downstream of retromer in their endosome recruitment (Figure 2H), our findings did not support the proposed role for RAB7A. Endogenous RAB7A did not co-immunoprecipitate with VPS35-FLAG expressed in VPS35-null cells (Figure S4E) nor was it enriched in the VPS35 interactome (Figure 2B-D and Table S1), and in a reverse immunoprecipitation experiment neither VPS35-FLAG or HA-TBC1D5 co-immunoprecipitated with GFP-RAB7A (Figure S4F). Consistent with these findings only a small amount of VPS35 overlapped with GFP-RAB7A⁺ puncta (Figure S4G and S4H). While AZD8055 and EBSS led to fewer VPS35⁺ vesicles, the overlap between VPS35 and RAB7A was unchanged by AZD8055 compared with the untreated condition but increased with EBSS (Figure S4G and S4H). Given that chemical inhibition of MTOR did not impede VPS35 recruitment to RAB7A⁺ endosomes, we surmise that the catalytic GAP activity of TBC1D5 was unlikely to be affected.

MTORC1 supports the trafficking of retromer cargoes

We speculated that MTORC1-dependent regulation of retromer may control the fate of cargo receptors trafficked from endosomes to the trans-Golgi network and plasma membrane. ADRB2/β₂ adrenergic receptor contains a PDZ binding motif within its C terminus that is essential for endosome-to-plasma membrane trafficking by SNX27-retromer (Figure S5A) [6]. Following transient expression HA-FLAG-tagged ADRB2 predominantly localized to the plasma membrane but redistributed to tubular and vesicular compartments throughout the cytoplasm following endocytosis induced by its agonist isoproterenol (Figure 5A) [6]. Depletion of VPS35 with siRNA increased overlap of HA-FLAG-ADRB2 on LAMP1⁺ vesicles following exposure to isoproterenol (Figure 5B,C). Similarly, MTOR inhibition with AZD8055 moderately increased the overlap of HA-FLAG-ADRB2 on LAMP1⁺ vesicles (Figure 5D,E), and concomitantly reduced its overlap with VPS35⁺ vesicles (Figure 5F,G). In a pulse-chase experiment, AZD8055 reduced the frequency of cells that recycled HA-FLAG-ADRB2 back to the plasma membrane following agonist-induced endocytosis in transfected HEK293T cells (Figure 5H). This indicated that MTORC1 supports an endosome-to-plasma membrane trafficking itinerary of retromer cargoes by preventing their turnover. To distinguish whether the endosome-to-Golgi trafficking function of retromer was also regulated by MTORC1 we assessed lysosomal turnover of the amyloid-β precursor protein (APP) [48], an established endosome-to-Golgi retromer cargo [58,59]. In live HeLa cells, both AZD8055 and EBSS augmented delivery of mCherry-GFP-APP to lysosomes as indicated by an increased mCherry:GFP signal (Figure S5B) [48]. Importantly, knock-down of the ESCRT-0 and -I components HGS and TSG101, respectively, did not inhibit starvation-dependent delivery of mCherry-GFP-APP to lysosomes suggesting it occurred independent of intraluminal vesicle/multivesicular body formation (Figure S5C-F). Moreover, in SH-SY5Y neuroblastoma cells, small vesicles containing endogenous APP decorated larger LC3B⁺ compartments following co-treatment with AZD8055 and BafA (Figure S5G and S5H). Taken together, we concluded that MTORC1 supports diverse receptor trafficking itineraries of retromer cargoes.

Figure 5. — MTORC1 supports the trafficking of retromer cargoes. (A) the agonist isoproterenol stimulates ADRB2 endocytosis. Cells transfected with HA-FLAG-ADRB2 were treated with or without isoproterenol for 1 h prior to immunofluorescence with a FLAG antibody. Blue, DAPI stained nuclei. Scale bar: 10 µm. Insets show 2.5× magnification. (B) ADRB2 accumulates within lysosomes in cells lacking VPS35. Cells transfected with HA-FLAG-ADRB2 were treated with *VPS35* or non-targeting control siRnas. Cells were treated with BafA for 2 h and isoproterenol for the final 30 min prior to immunofluorescence with a FLAG and LAMP1 antibodies. Blue, DAPI stained nuclei. Scale bar: 10 µm. Insets show 2.5× magnification. (C) HA-FLAG-ADRB2 colocalization with LAMP1 from (B). Values are the mean ± SEM from n = 6–8 cells/condition (unpaired t-test). (D) ADRB2 accumulates moderately within lysosomes following MTOR inhibition. Cells transfected with HA-FLAG-ADRB2 treated with or without AZD8055 for 6 h. Cells were treated with leupeptin and pepstatin (LP) for the final 4 h and isoproterenol for the final 1 h to immunofluorescence with FLAG and LAMP1 antibodies. Blue, DAPI stained nuclei. Scale bar: 10 µm. Insets show 2.5× magnification. (E) HA-FLAG-ADRB2 colocalization with LAMP1 from (D). Values are the mean ± SEM from n = 26–31 fields/condition from three independent experiments (unpaired t-test). (F) reduced association of ADRB2 with retromer following MTOR inhibition. Cells transfected with HA-FLAG-ADRB2 treated with or without AZD8055 for 6 h, and isoproterenol for the final 1 h prior to immunofluorescence with FLAG and VPS35 antibodies. Blue, DAPI stained nuclei. Scale bar: 10 µm. Insets show 2.5× magnification. (G) HA-FLAG-ADRB2 colocalization with VPS35 from (F). Values are the mean ± SEM from n = 25 fields/condition from three independent experiments (unpaired t-test). (H) MTOR inhibition impairs HA-FLAG-ADRB2 recycling back to the plasma membrane following agonist-induced endocytosis. Cells transfected with HA-FLAG-ADRB2 receptor were treated with or without AZD8055 for 6 h, then were pulsed with isoproterenol for 30 min to induce endocytosis and then chased for 10 min to allow recycling to occur. Cell surface HA-FLAG-ADRB2 was measured by flow cytometry. Recycling index refers to the frequency of transfected cells that recycled the receptor back to the cell surface. Values are the mean ± SEM from three independent experiments (One-sample t and Wilcoxon test compared to 1). (I) model showing how nutrients (such as amino acids) activate MTORC1 to inhibit capture of retromer-TBC1D5 decorated endosomes by autophagosomes to promote receptor recycling.

Discussion

Our findings support a mechanism that inhibits receptor recycling by coordinating TBC1D5-dependent capture of retromer⁺ endosomes by autophagosomes for wholesale destruction (Figure 5I). While this targeting to autophagosomes exhibited a preference for retromer⁺ endosomes and was strongly regulated by nutrient withdrawal and MTORC1 inhibition, our work raises several interesting questions.

Does this represent a bona-fide selective autophagy pathway for endosomes (i.e., endophagy), or instead, nonselective turnover of endosomes via macroautophagy? If it were truly selective, the signals which occur at endosomes to coordinate this process are yet to be revealed. Our initial hypothesis was that TBC1D5 may serve as a selective autophagy receptor for retromer⁺ endosomes. However, TBC1D5 did not satisfy criteria for this to be true. The C-terminal LIR #3 of TBC1D5 was not necessary for autophagosomes to capture retromer⁺ endosomes, and TBC1D5 did not interact with ATG8 proteins in our experiments.

Why do retromer⁺ endosomes appear to be preferentially targeted to autophagosomes for destruction? One possible explanation may be related to their ability to specifically recruit TBC1D5 to endosomes, given the importance of TBC1D5 in this process. Additionally, retromer may serve as a “master substrate” because its turnover likely affects numerous receptor trafficking events in parallel [11]. Interestingly, the total cellular amount of retromer components were largely unchanged by nutrient withdrawal or MTOR inhibition, indicating that a small – likely endosome-associated and active – pool of retromer is targeted for destruction [55]. Consistent with this, only a small fraction of retromer in the cell is associated with endosomal membranes. Alternatively, or in parallel, retromer turnover may also be compensated by TFEB-driven transcription of its components [60]. Moreover, specific coordination of retromer by MTORC1 and autophagy may synchronize various receptor-mediated signaling events with nutrient availability and other environmental cues. Similarly, amino acids were demonstrated to support endosome-to-Golgi trafficking of the IGF2R and SORT1, which extends our observations to additional retromer cargoes [61]. A key difference was that amino acids achieved this through the Ragulator-SLC38A9 system (which controls MTORC1 activation) independently of MTORC1 activity, indicating that receptor homeostasis may be regulated in parallel at different points along the MTORC1 pathway [61]. Moreover, stress-induced phosphorylation of the retromer-adaptor SNX27 by MAPK11-MAPK14 was found to inhibit cargo selection and receptor recycling to the plasma membrane [36]. This suggests that multiple mechanisms may act in parallel or potentially in concert to suppress receptor recycling by retromer when nutrients are limiting. Interestingly, retromer deficiency perturbs MTORC1 activation by amino acids [27], and confers insensitivity to rapamycin in yeast [62], suggesting potential feedback between these pathways.

Moreover, nutrient-responsive capture of retromer⁺ endosomes by autophagosomes appears to be distinct from amphisome formation (i.e., because retromer was membrane-protected, colocalized with nonacidic LC3B⁺ compartments, and its cargoes were delivered to lysosomes in an ESCRT-independent fashion). During nutrient stress, wholesale capture of endosomes via autophagosomes likely serves to deliver both their membrane (e.g., retromer itself and other cargo receptors) and luminal contents to lysosomes for destruction to salvage nutrients. This is analogous to observations made during the early phase of starvation in yeast [63]. To achieve the same result via amphisome formation, retromer⁺ endosomes would first have to fuse with autophagosomes, then additional membrane invagination events would need to occur to internalize membrane contents such as the retromer-TBC1D5 complex to ensure their turnover by lysosomes.

Because retromer is captured by autophagosomes in a TBC1D5-dependent manner following MTORC1 inhibition, it is tempting to speculate that this may also occur in response to other stimuli. Consistent with this, retromer associates with LC3B⁺ compartments following hepatitis C virus infection [64], and its components VPS35 and VPS26 comprise part of the autophagy degradome [65,66]. Interestingly, following human cytomegalovirus infection, the viral protein M45 targets IKBKG/NEMO for autophagic clearance in a VPS26B- and TBC1D5-dependent manner to suppress NFKB (nuclear factor kappa B) activation and inflammatory signaling [67]. Moreover, while our data suggest that retromer localizes to the inside of autophagosomes we cannot exclude the possibility that, in parallel, a distinct pool of retromer may localize to the outside of autophagosomes where it may exhibit a similar receptor sorting function, akin to the recently described recycler complex [68].

A previous model of retromer regulation indicated that during autophagy following glucose starvation, TBC1D5 disassociates from retromer because it prefers to interact with ATG8 proteins on autophagosomes through its C-terminal LIR #3 [35,37–39]. This is thought to increase RAB7A^GTP-mediated recruitment of retromer to endosomes – as TBC1D5 is a GAP for RAB7A – to increase recycling of receptors such as the glucose transporter SLC2A1 [13,35,37–39,69]. For clarity, we will henceforth refer to this as the “previous model” of retromer regulation. Our findings deviate from the previous model and provide an alternative model of retromer regulation. We found that during autophagy triggered by MTOR inhibition, the retromer-TBC1D5 complex was targeted to autophagosomes for destruction independently of its C-terminal LIR^#3 and this bulk endosomal turnover inhibited receptor recycling.

Key differences between the previous model and ours are as follows: (1) we failed to identify an interaction between TBC1D5 or retromer with any ATG8 family members and neither starvation nor MTOR inhibition abrogated the TBC1D5-retromer interaction. It is important to consider that almost all examples of TBC1D5-ATG8 interactions, which predicate the previous model, employed purified proteins for in vitro binding assays and GST-pulldowns, experimental approaches that do not faithfully recapitulate endogenous stoichiometry, stable binding partners, or other complexities such as post-translation modifications [35]. In contrast, co-immunoprecipitation experiments from cell cultures relied on overexpression or failed to control for non-specific interactions [35–37]. Notably, interactions between TBC1D5 and GFP-LC3B, as well as ULK1 [39] were artefactual in our hands. Further, the study that identified TBC1D5-ATG8 interactions using in vitro binding assays and overexpression of both proteins in cells failed to identify any ATG8 family members in their assessment of the TBC1D5 interactome [35]. In addition, these proteins do not appear as experimentally determined interactors in either HEK293T or HCT116 cells from the Bioplex 3.0 database [70]. One possibility is that TBC1D5-ATG8 interactions may only exist for the short duration of autophagosome flux before fusion with lysosomes which may limit their detection [71]. However, this is unlikely to be the case because the lysosome inhibitor BafA did not stabilize such interaction in our hands. Further, while the C-terminal LIR #3 of TBC1D5 was not involved in targeting retromer to autophagosomes, we cannot exclude the possibility that LIR #1 or LIR #2 may be involved despite our inability to demonstrate an interaction between wild-type TBC1D5 and ATG8 proteins. Because VPS29 and ATG8 proteins compete for binding to LIR #1 of TBC1D5 in vitro [35], our data argue against involvement of this LIR given that the TBC1D5-retromer interaction was not affected by MTOR inhibition or nutrient withdrawal.

It was previously reported that RAB7A^GTP binds retromer and recruits it to the endosomal surface where receptor sorting and recycling occurs, before RAB7A^GTP undergoes hydrolysis by TBC1D5 to displace retromer from endosomes [13]. In disagreement with these findings, (2) we found that most retromer⁺ vesicles were not RAB7A⁺, and that retromer did not interact with RAB7A. Further, retromer recruitment to RAB7A⁺ endosomes was not impacted by autophagy via MTOR inhibition indicating that RAB7A^GTP hydrolysis by TBC1D5 was unlikely to be stimulated. This interpretation, however, relied on the assumption that retromer is a RAB7A effector.

Finally, (3) the previous model centred around glucose deprivation as the autophagy stimulus, which augmented trafficking of the retromer cargo SLC2A1 to the plasma membrane to enhance glucose uptake [37,38]. In contrast, our study focused on autophagy induced by either nutrient stress or catalytic inhibition of MTOR which impaired the trafficking of retromer cargoes to the plasma membrane (e.g., ADRB2) and Golgi (e.g., APP). Importantly, autophagy activation by MTORC1 inhibition with rapamycin did not phenocopy the effect of glucose deprivation on SLC2A1 trafficking [37]. Inconsistent with the previous model, a VPS29^L152E mutant that failed to bind TBC1D5 did not increase SLC2A1 recycling but rather reduced it [5,72]. Thus, it is plausible that the coordination of retromer by autophagy may have cargo-specific effects or may depend on which nutrients are limiting. Indeed, nutrient stress was recently reported to increase SLC2A1 delivery to lysosomes for degradation [36], the opposite of what was reported to occur during glucose deprivation [37].

In summary, we reveal a mechanism where nutrient insufficiency increases receptor degradation by coordinating capture of retromer⁺ endosomes by autophagosomes in a TBC1D5-dependent fashion. These findings provide additional insights about how retromer function is antagonized by TBC1D5.

Materials and methods

Materials used in this study are listed in Table 1.

Table 1.

Materials used in this study.

Resource	Source	Identifier
Antibodies
Donkey anti-Goat IgG (H+L)-AF647	ThermoFisher	A32849
Donkey anti-Goat IgG (H+L)-AF488	Jackson Immuno Research Laboratory	705-545-003
Donkey anti-Goat IgG (H+L)-AF594	Jackson Immuno Research Laboratory	705-585-003
Donkey anti-Mouse IgG (H+L)-Cy3	Jackson Immuno Research Laboratory	715-165-150
Donkey anti-Rabbit IgG (H+L)-Cy3	Jackson Immuno Research Laboratory	711-165-152
Donkey polyclonal anti-Sheep/Goat IgG (H+L)-HRP	Millipore	AB324P
Goat polyclonal anti-Mouse IgG (H+L)-HRP	Bio-Rad	170–6516
Goat polyclonal anti-Rabbit IgG (H+L)-HRP	Millipore	AP307P
Goat polyclonal anti-VPS35	Abcam	Ab10099
Mouse monoclonal anti-ACTB/Beta Actin-HRP	Sigma-Aldrich	A3854
Mouse monoclonal anti-RPS6	ThermoFisher	MA5–15123
Mouse monoclonal anti-FLAG M2	Sigma-Aldrich	F1804
Mouse monoclonal anti-APP (6E10)	BioLegend	803001
Rabbit monoclonal anti-FLAG (D6W5B)	Cell Signalling Technology	14793
Mouse monoclonal anti-HA.11 (16B12)	Biolegend	901513
Mouse monoclonal anti-TSG101 (4A10)	Abcam	Ab83
Mouse monoclonal anti-EEA1 (G4)	Santa Cruz Biotechnology	sc137130
Rabbit polyclonal anti-HA	Sigma-Aldrich	SAB4300603
Rabbit monoclonal anti-HA (C29F4)	Cell Signalling Technology	3724
Goat polyclonal anti-GFP	Rockland	600-101-215
Rabbit polyclonal anti-GFP	Invitrogen	A6455
Mouse monoclonal anti-GAPDH	Sigma-Aldrich	G8795
Mouse monoclonal anti-LAMP2	In house	Validated in [73]
Rabbit monoclonal anti-ATG5 (clone D5F5U)	Cell Signalling Technology	12994
Rabbit monoclonal anti-RPTOR	Cell Signalling Technology	24C12
Rabbit monoclonal anti-RRAGC	Cell Signalling Technology	D31G9
Rabbit monoclonal anti-NPRL2	Cell Signalling Technology	37344
Rabbit monoclonal anti-MIOS	Cell Signalling Technology	13557
Rabbit monoclonal anti-VPS26	Abcam	Ab181352
Rabbit polyclonal anti-VDAC	Cell Signalling Technology	4866
Rabbit polyclonal anti-RPS6 (P-S235/236)	Cell Signalling Technology	2211
Rabbit polyclonal anti-LC3B	Novus	NB100–2220
Rabbit polyclonal anti-LC3B (clone D11)	Cell Signalling Technology	3868
Rabbit polyclonal anti-LC3A	Abcam	Ab62720
Rabbit polyclonal anti-GABARAP	ThermoFisher	PA588628
Rabbit polyclonal anti-VPS29	Abcam	Ab98929
Rabbit polyclonal anti-RPS6 (P-S240/244)	Cell Signalling Technology	2215
Rabbit monoclonal anti-ULK1	Cell Signalling Technology	8054
Rabbit polyclonal anti-ULK1 (P-S758)	Cell Signalling Technology	6888
Rabbit polyclonal anti-ULK1 (P-S555)	Cell Signalling Technology	5869
Rabbit polyclonal anti-HGS	ThermoFisher	PA5–27491
Rabbit monoclonal anti-AKT (clone 11E7)	Cell Signalling Technology	4685
Rabbit polyclonal anti-AKT (P-S473)	Cell Signalling Technology	9271
Rabbit polyclonal anti-RPS6KB1/S6K1	Cell Signalling Technology	9202
Rabbit polyclonal anti-RPS6KB1/S6K1 (P-T389)	Cell Signalling Technology	9205
Rabbit polyclonal anti-TBC1D5	Proteintech	17078–1-AP
Rabbit monoclonal anti-RAB7 (clone D95FU)	Cell Signalling Technology	9367
Rabbit monoclonal anti-TAX1BP1 (clone D1D5)	Cell Signalling Technology	5105
Rabbit polyclonal anti-SQSTM1	MBL	PM045
Cell culture reagents
HeLa cells	Sigma-Aldrich	93021013-1VL
HEK293T	Takara	632180
SH-SY5Y	ATCC	CRL-2266
Earle’s balanced salt solution (EBSS) containing calcium, magnesium and phenol red	ThermoFisher	24010043
Dulbecco’s phosphate buffered saline (DPBS)	ThermoFisher	14190144
Fetal bovine serum (FBS)	ThermoFisher	10099141
One Shot Dialyzed FBS	ThermoFisher	A3160601
Gibco Dulbecco’s minimum essential medium (DMEM) containing high glucose and L-glutamine	ThermoFisher	11965092
DMEM for SILAC containing high glucose and L-glutamine without Arginine and Lysine	ThermoFisher	88364
DMEM/F12 + GlutaMAX with sodium bicarbonate and sodium pyruvate	ThermoFisher	10565–018
Lipofectamine 2000	ThermoFisher	11668027
Lipofectamine RNAi Max Transfection Reagent	ThermoFisher	13778150
Opti-MEM	ThermoFisher	31985070
Gibco 100× MEM essential amino acids (without L-glutamine)	ThermoFisher	11130–051
Gibco 100× MEM non-essential amino acids	ThermoFisher	11140–050
L-glutamine solution (200 mM)	Sigma-Aldrich	59202C
Drugs
AZD8055	Selleckchem	S1555
Alprenolol hydrochloride	Selleckchem	S5802
BafA	Selleckchem	S1413
CQ diphosphate salt	Sigma-Aldrich	C6628
Isoproterenol hydrochloride	Sigma-Aldrich	I6504
LY2584702	Selleckchem	S7698
Rapamycin	Selleckchem	S1039
Leupeptin hemisulfate salt	Sigma-Aldrich	L8511
Pepstatin A	Sigma-Aldrich	P4265
Reagents
Ampicillin sodium salt	Sigma-Aldrich	A9518
BamHI-HF	New England Biolabs	R3136S
BES	Sigma-Aldrich	B9879
Cutsmart buffer	New England Biolabs	B7204S
Quick CIP	New England Biolabs	M0525S
T4 polynucleotide kinase	New England Biolabs	M0201S
T4 ligase	New England Biolabs	M0202S
T4 ligase buffer	New England Biolabs	B0202S
10× Bolt Sample Reducing Agent	ThermoFisher	B0009
BCA protein assay kit	ThermoFisher	23225
β-glycerophosphate	Sigma-Aldrich	G9422
BOLT 4–12% Bis-Tris Plus Gels, 10 well	ThermoFisher	NW04120BOX
BOLT 4–12% Bis-Tris Plus Gels, 12 well	ThermoFisher	NW04125BOX
BOLT 12% Bis-Tris Plus Gel, 15 well	ThermoFisher	NW00120BOX
Bovine serum albumin	Sigma-Aldrich	A9647
cOmplete, EDTA-free protease inhibitor cocktail	Roche	04693132001
DH5 alpha competent E. coli (high efficiency)	New England Biolabs	C2987I
Stable competent E. coli (high efficiency)	New England Biolabs	C3040H
Glycine	Millipore	VP709001610
Formaldehyde solution, 10% neutral buffered	LabServ	BSPFS426.2.5
Ponceau S stain	Sigma-Aldrich	P7170
Precision Plus Protein Kaleidoscope standard	Bio-Rad	1610375
Anti-FLAG M2 magnetic beads	Sigma-Aldrich	M8823
Protein G Dynabeads	ThermoFisher	10003D
Pierce^TM Anti-HA Magnetic beads	ThermoFisher	88837
Puromycin dichloride	Sigma-Aldrich	P8833
Saponin	Sigma-Aldrich	S4521
Sodium chloride	Chem-Supply Pty Ltd	SA046
Sodium fluoride	Sigma-Aldrich	S7920
Sodium orthovanadate	Sigma-Aldrich	S6508
Sodium pyrophosphate	Sigma-Aldrich	P8135
SuperSignal West Femto Maximum Sensitivity Substrate	ThermoFisher	34095
SuperSignal West Pico PLUS Chemiluminescent Substrate	ThermoFisher	34577
Tris base	Millipore	648310
Triton X-100	Sigma-Aldrich	X100-1 L
Tween-20	Millipore	S7679184848
Vectashield Hard Set Mounting Medium with DAPI	Vector Laboratories	H-1500
Low protein binding collection tubes (1.5 mL)	ThermoFisher	90410
Magnesium chloride	Sigma-Aldrich	M8266
HEPES	Sigma-Aldrich	H3375
Sodium azide	Sigma-Aldrich	S2002
Proteinase K	New England Biolabs	P8107S
Trichloroacetic acid	Sigma-Aldrich	T4885
MycoAlert^TM PLUS Mycoplasma Detection Kit	Lonza	LT-07-705
Normal Donkey Serum	Jackson Immuno Research Laboratory	017000121
Gibco Versene	ThermoFisher	15040–066
Recombinant DNA
HA-tag insert Forward oligo for FLAG-ADRB2 (5’-gatcaccggtATGTACCCATACGATGTTCCAGATTACGCTg-3’)	Integrated DNA Technologies	N/A
HA-tag insert reverse oligo for FLAG-ADRB2 (5’-gatccAGCGTAATCTGGAACATCGTATGGGTACATaccggt-3’)	Integrated DNA Technologies	N/A
Negative control silencer select siRNA oligonucleotide #1 (siRNA ID: 4390843)	ThermoFisher	4427037
Negative control silencer select siRNA oligonucleotide #2 (siRNA ID: 4390846)	ThermoFisher	4427037
pUltrahot	Addgene; gift from Malcom Moore	24130
pUltrahot-VPS35-FLAG	Previous study [30]	N/A
pUltrahot-GFP	Previous study [30]	N/A
pJMC-3×HA-GST	This study	N/A
pJMC-3×HA-TBC1D5	This study	N/A
pJMC-3×HA-TBC1D5 (S560A/S561A/S566A)	This study	N/A
pJMC-3×HA-TBC1D5 (S560D/S561D/S566D)	This study	N/A
pJMC-3×HA-TBC1D5 (L142E)	This study	N/A
pJMC-3×HA-TBC1D5 (F809A/V812A)	This study	N/A
pcDNA3.1(+)	Gift from Christopher Proud	N/A
pcDNA3-FLAG-ADRB2	Addgene; gift from Robert Lefkowitz [74]	14697
pcDNA3-HA-FLAG-ADRB2	This study	N/A
pRK5-Rheb Y35N-FLAG	Gift from Christopher Proud	N/A
pAcGFP1-C1- RAB7A	Addgene; gift from Gia Voeltz [75]	61803
pUC19-sgTBC1D5 #1 (pPN144)	Addgene; gift from Lindy Barrett [76]	91673
pUC19-sgTBC1D5 #2 (pPN145)	Addgene; gift from Lindy Barrett [76]	91674
pLentiCRISPR v2	Addgene; gift from Feng Zhang [77]	52961
pCMV-VSV-G	Addgene; gift from Bob Weinberg [78]	8454
psPAX2	Addgene; gift from Didier Trono	12260
VPS35 silencer select siRNA oligonucleotide #1 (siRNA ID: s31374)	ThermoFisher	4427037
VPS35 silencer select siRNA oligonucleotide #2 (siRNA ID: s31375)	ThermoFisher	4427037
HGS silencer select siRNA oligonucleotide #1 (siRNA ID: s17480)	ThermoFisher	4427037
HGS silencer select siRNA oligonucleotide #2 (siRNA ID: s17481)	ThermoFisher	4427037
HGS silencer select siRNA oligonucleotide #3 (siRNA ID: s17482)	ThermoFisher	4427037
TSG101 silencer select siRNA oligonucleotide #1 (siRNA ID: s14439)	ThermoFisher	4427037
TSG101 silencer select siRNA oligonucleotide #2 (siRNA ID: s14440)	ThermoFisher	4427037
TSG101 silencer select siRNA oligonucleotide #1 (siRNA ID: s14441)	ThermoFisher	4427037
Limma R package	Bioconductor, v3.4.2	https://bioconductor.org/packages/release/bioc/html/limma.html
R	R Studio, v3.6.1	https://rstudio.com/products/rpackages/
MaxQuant software	MaxQuant, v1.6.10.43	https://www.maxquant.org
iLIR database	iLIR database	https://ilir.warwick.ac.uk [57],
Bioplex 3.0 database	Bioplex 3.0 database	https://bioplex.hms.harvard.edu [70]
Chimera X	UCSF, v1.2.5	https://www.cgl.ucsf.edu/chimerax/ [79]
PONDR-Fit	PONDR-Fit	http://www.pondr.com, [80]
AlphaFold	AlphaFold	https://alphafold.ebi.ac.uk [81]
PhosphoSitePlus	CST, v6.5.9.3	https://www.phosphosite.org/homeAction.action
Reference human proteome with isoforms	Uniprot, downloaded March 2019	https://www.uniprot.org
Illustrator	Adobe	https://www.adobe.com/au/products/illustrator.html

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Molecular cloning

pJMC vectors were derived by removing mCherry-P2A-T2A cDNA from pUltrahot by restriction digest. cDNAs encoding 3×HA-GST, or TBC1D5 and variants were manufactured as gBlocks and underwent restriction cloning into pJMC vectors. Plasmids were propagated under antibiotic selection in DH5 alpha competent E. coli or stable competent E. coli, and were validated by Sanger sequencing.

Cell culture

HeLa and HEK293T cells were maintained in DMEM containing high glucose and L-glutamine supplemented with 10% FBS; SH-SY5Y cells were maintained in DMEM/F12 + GlutaMAX with sodium bicarbonate and sodium pyruvate supplemented with 10% FBS. All cells were cultured at 37°C with 5% CO₂ and were routinely screened for mycoplasma contamination. RFP-GFP-LC3B expressing, mCherry-GFP-APP expressing, ATG5-null, VPS35-null, and VPS35-FLAG rescue HeLa cells have been described elsewhere [19,26,30,48]. Stable expression of HA-GST or HA-TBC1D5 and variants in TBC1D5-null and VPS35-FLAG-expressing VPS35-null HeLa cells, or FLAG-mTurbo-LC3B in HeLa cells was achieved by lentiviral infection as previously described [30].

CRISPR-Cas9 gene editing

To delete TBC1D5, HeLa cells were co-transfected with a pUC19 vector encoding the sgRNA and empty pLentiCRISPRv2 encoding Cas9-P2A-PuroR (3:1 ratio). sgRNAs targeting exon 4 of TBC1D5 were employed (sgTBC1D5 #1: 5-ATGCCTACTTCTTGTTCTTC-3; and sgTBC1D5 #2: 5’-TCCAGTTACTCTAACAAGTC-3’). Two days later, cells were re-plated in culture medium containing 1 µg/mL puromycin for two days (i.e., when negative control cells were dead) then allowed to recover for one week. Single cell-derived colonies were expanded and screened for the absence of TBC1D5 protein by immunoblotting and immunofluorescence.

Transfection

For cDNA expression in HeLa cells, cells were plated to achieve ~ 60–70% confluence the following day and transfected with plasmids using Lipofectamine 2000, according to the manufacturer’s instructions. Treatments/harvests were carried out ~48 h later.

For cDNA expression in HEK293T cells, cells were plated to achieve ~ 20% confluence the following day and transfected with plasmids using the BES-calcium chloride method [82] in serum-free culture medium for 4 h before the re-addition of culture medium containing serum. Treatments/harvests were carried out ~72 h later.

For knockdowns, cells were plated to achieve ~ 30% confluence the following day during reverse transfection with 30 pmol siRNA oligonucleotides and Lipofectamine RNAi Max, according to the manufacturer’s instructions. Two rounds of reverse transfections were performed for a five-day exposure to siRNAs for VPS35 as it is a long-lived protein. Cells were analyzed at least 48 h after the final reverse transfection.

Drug treatments

Drugs were diluted into culture medium and incubated with cells for the indicated times. Drugs were used at the following final concentrations: 1 µM AZD8055; 100 nM rapamycin; 200 nM bafilomycin A1 (BafA), 50 µM chloroquine (CQ); 10 µM isoproterenol; 10 µM alprenolol and 50 µg/mL leupeptin and 4 µM pepstatin which were used in combination (LP).

Starvation experiments

For serum and amino acid starvation, cells were washed twice with DPBS then maintained in EBSS for the times indicated. For comparisons between EBSS and full medium containing vehicle or AZD8055, vehicle was added to the EBSS condition.

For serum and amino acid starvation/re-stimulation, cells were washed twice with DPBS and starved in EBSS for 1 h. Following this, cells were treated with EBSS supplemented with 1× essential amino acids, 1× non-essential amino acids, 2 mM L-glutamine, and 10% dialyzed FBS for the times indicated up to 1 h. For the un-starved condition, cells were maintained in EBSS supplemented with 1× essential amino acids, 1× non-essential amino acids, 2 mM L-glutamine, and 10% dialyzed FBS for 2 h.

For arginine and lysine deprivation, cells were washed twice with DPBS and starved in DMEM containing high glucose, and L-glutamine lacking arginine and lysine supplemented with 10% dialyzed FBS for the times indicated up to 8 h. For the un-starved condition, cells were maintained in DMEM containing high glucose and L-glutamine supplemented with 10% dialyzed FBS for 8 h.

Immunofluorescence, confocal microscopy, and quantification

Cells were plated on coverslips and maintained at 37°C until harvest. Cells were washed twice with DPBS and fixed with 10% neutral buffered formaldehyde for 15 min. Fixed cells were permeabilized with 0.1% saponin in DPBS for 15 min or, alternatively, were simultaneously fixed and permeabilized with ice-cold methanol for 15 min at −20°C to detect endogenous LC3B or TBC1D5. Cells were blocked with 3% bovine serum albumin, 0.01% saponin in DPBS for 1 h. Cells were immunostained with primary antibodies diluted in block overnight at 4°C and then washed four-times with DPBS. Fluorophore-conjugated secondary antibodies were diluted 1:500 in block and used to immunostain cells for 1 h in the dark prior to washing four-times with DPBS. Coverslips were mounted onto glass slides with mounting medium containing DAPI and sealed with clear nail polish. Slides were kept in the dark at 4°C until confocal microscopy analysis. Images or z-stacks were captured at 63× or 40× objective using a TCS SP8X multi-photon confocal microscope with LASX software (Leica, Germany) using identical acquisition conditions between treatment groups. Images were processed and quantified using FIJI ImageJ.

Analysis of puncta per cell:

z-stacks were max-projected, and the channel of interest was “threshold”-ed, made “binary” and “watershed”-ed. Regions of interest were drawn around individual cells in an unmodified duplicate of the field, and its coordinates were saved in the “ROI manager” before being superimposed onto the binary image. For each region of interest/cell “analyze particles” was used to calculate the number of puncta (>0.05 μm²). Alternatively, the number of puncta per cell was calculated from a field of view containing numerous cells and normalized to DAPI-positive cell number.

Colocalization analysis:

single z-plane channels were “threshold”-ed using the JaCOP plugin, which was used to calculate the colocalization index (Mander’s co-efficient) (Figure 5 and S4) [83].

Alternatively, single z-plane channels were “auto threshold”-ed using the “moments” method (“moments” was empirically determined to be the most accurate auto threshold method using pilot data). From these, the “image calculator” function was used to generate a binary image of particles present in both the binary VPS35 “AND” LC3B channels (i.e., double-positive). “Analyze particles” was used to quantify the positive area (>0.05 μm²) for each channel, and double-positive signal was expressed as a fraction of total VPS35 signal (Figure 4). A similar approach was used for EEA1, VPS26 and FLAG-LC3B (Figure 3), and APP and LC3B colocalization (Figure S5).

To calculate the number of autophagosomes (GFP⁺ and RFP⁺) and autolysosomes/amphisomes (GFP⁻ and RFP⁺) from HeLa cells that stably express RFP-GFP-LC3B, single z-plane channels were “auto threshold”-ed and made binary. The number of autophagic vesicles (autophagosomes and autolysosomes/amphisomes) (>0.1 μm²) were calculated using “Analyze particles” on the RFP channel. The number of autophagosomes (>0.1 μm²) were calculated using “Analyze particles” on the GFP channel. The number of autolysosomes/amphisomes was calculated by subtracting the number autophagosomes from the number of autophagic vesicles (Figure 3C). To calculate the number of VPS35⁺ autophagosomes, the “image calculator” function was used to generate a binary image of particles present in both the binary VPS35 “AND” GFP channels (i.e., double-positive). “Analyze particles” was used to quantify the number of double-positive signals (>0.1 μm²) (Figure 3D).

Cell lysis and immunoprecipitation

Cells were washed twice with ice-cold DPBS then lysed on ice for 20 min with lysis buffer containing 1% Triton X-100, 40 mM HEPES, pH 7.4, 2.5 mM MgCl₂, phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 50 mM sodium fluoride) and EDTA-free protease inhibitor cocktail (1 tablet/25 mL buffer). Cells were then collected and clarified at 21,000 × g for 10 min at 4°C. A well-mixed 50% slurry of either anti-FLAG M2-, anti-HA- or antibody-bound protein G-magnetic beads were added to low protein binding tubes and washed three-times with incomplete lysis buffer lacking phosphatase and protease inhibitors. Cleared lysate was incubated with the beads for 2 h at 4°C. The beads were washed three-times, once with complete lysis buffer and twice with complete lysis buffer containing 500 mM NaCl. Immunoprecipitated proteins were eluted by boiling in 2×LDS sample buffer for 5 min at 95°C for immunoblotting or eluted in 50 mM glycine (pH 4) for 10 min at room temperature before neutralization with 1 M Tris, pH 8 for relative label-free quantitation proteomics.

Immunoblotting

Cleared lysates were combined with 1× LDS sample buffer and 1× reducing agent prior to boiling for 5 min at 95°C, then loaded onto pre-cast gels and run at 130 V for up to 90 min. Total protein was transferred to methanol-activated PVDF membranes at 35 V for 90 min. Membranes were blocked for 1 h with 5% fat-free skim milk in TBST (50 mM Tris, 150 mM sodium chloride, 0.1% Tween-20) and then probed with primary antibodies diluted in 2% bovine serum albumin in TBST containing 0.02% sodium azide, with rotation overnight at 4°C. Membranes were washed three-times with TBST then re-probed with HRP-conjugated secondary antibody diluted 1:10,000 in block. Membranes were washed a further three-times then imaged by chemiluminescence using a ImageQuant LAS4000 luminescent image analyzer (GE Healthcare). Quantification of densitometry was performed using FIJI ImageJ.

Relative label-free quantitation proteomics

Eluates were resuspended in 6 M urea, 10 mM TCEP, and 100 mM Tris-HCl, pH 7.0 and subjected to protein digestion using FASP (filter aided sample preparation) before lyophilization to dryness using a SpeedVac AES 1010 (Savant, ThermoFisher) [84]. Samples were analyzed on an M-Class UHPLC (Waters, USA) coupled to a timsTOF Pro (Bruker) mass spectrometer equipped with a CaptiveSpray source. Peptides were resuspended in 2% acetonitrile, 1% formic acid separated on a 25 cm × 75 μm analytical column, 1.6 μm C18 beads with a packed emitter tip (IonOpticks, AUR2-25075C18A). The column temperature was maintained at 40°C using an integrated column oven (Sonation GmbH, Germany). The column was equilibrated using five column volumes before loading sample in 100% buffer A (0.1% formic acid). Samples were separated at 400 nL/min using a gradient from 2% to 17% buffer B (99.9% acetonitrile, 0.1% formic acid; 55 min), 17% to 25% buffer B (21 min) before ramping to 35% buffer B (13 min), then to 85% buffer B (3 min), and sustained for 10 min. The timsTOF Pro (Bruker) was operated in PASEF mode using Compass Hystar 5.0.36.0. Settings were as follows: Mass Range 100 to 1700 m/z, 1/K0 Start 0.6 V.s/cm² End 1.6 V.s/cm²; Ramp time 109.9 ms; Lock Duty Cycle to 100%; Capillary Voltage 1600 V; Dry Gas 3 l/min; Dry Temp 180°C; PASEF settings: 10 MS/MS scans (total cycle time 1.26 s); charge range 0–5; active exclusion for 0.4 min; Scheduling Target intensity 20,000; Intensity threshold 2500; CID collision energy 42 eV. All raw files were analyzed by MaxQuant software using the integrated Andromeda search engine. Experiment type was set as TIMS-DDA with no modification to the default settings. Data were searched against the human Uniprot Reference Proteome with isoforms and a separate reverse decoy database using a strict trypsin specificity allowing up to two missed cleavages. The minimum required peptide length was set to seven amino acids. Modifications: Carbamidomethylation of Cys was set as a fixed modification; N-terminal acetylation of proteins, oxidation of M, and phosphorylation of S/T/Y were set as variable modifications. First search peptide tolerance was set at 20 ppm and main search set at 6 ppm (other settings left as default). Matching between runs and relative label-free quantitation was turned on. Maximum peptide mass was set at 8000 Da. All other settings in group or global parameters were left as default. Further analysis was performed using a custom pipeline developed in R, which utilized the label-free quantitation intensity values in the MaxQuant output file proteinGroups.txt. Proteins not found in at least 50% of the replicates in one group were removed. Missing values were imputed using a random normal distribution of values, with the mean set at mean of the real distribution of values minus 1.8 standard deviation, and a standard deviation of 0.3× the standard deviation of the distribution of the measured intensities. The probability of differential site modification expression between groups was calculated using the Limma R package. P values were adjusted for multiple testing using Benjamini – Hochberg method.

Protease protection assay

Protease protection assays were performed as previously described but with minor modifications [54,85]. Cells were washed twice with ice-cold DPBS and collected by scraping into DPBS on ice. Each 15 cm dish of cells was homogenized with 20 strokes of a probe homogenizer on ice; the post-nuclear lysate was then collected following clarification at 500 × g for 5 min at 4°C. Equal amounts (by protein content) of post-nuclear lysate were divided across three tubes that received 50 μg/mL proteinase K with or without 0.2% Triton X-100 for 20 min on ice, or were left untreated. Reactions were stopped by the addition of 1 mM phenylmethylsulfonyl fluoride for 10 min on ice. Proteins were precipitated with trichloroacetic acid for 30 min on ice, and protein pellets were washed twice with acetone. Dried protein pellets were dissolved in 2× LDS sample buffer and 1× reducing agent prior to boiling for 5 min at 95°C, and analysis by immunoblotting.

Light-membrane and cytoplasm fractionation

HeLa cells were plated in a 10-cm dish. After ~48 h, cells were washed twice with ice-cold DPBS and collected by scraping into homogenization buffer (DPBS containing 10 mM Sodium Pyrophosphate, 10 mM Beta-Glycerophosphate and 1× EDTA-free protease inhibitor) on ice. Cells were homogenized with 20 strokes of a probe homogenizer on ice, then clarified at 500 × g for 5 min at 4°C to remove un-lysed cells, debris, and nuclei. The post-nuclear supernatant (400 uL) was subsequently centrifuged at 20,000 × g for 20 min at 4°C to pellet light-membranes. The supernatant was kept as the cytoplasm-enriched fraction, and the light-membrane pellet was washed with 400 uL of homogenization buffer and re-centrifuged at 20,000 × g for 5 min at 4°C. The pellet was resuspended in 400 uL of homogenization buffer and kept as the light-membrane-enriched fraction. Equal volumes of cytoplasm-enriched (supernatant) and light-membrane-enriched fraction (pellet) were combined with 1× LDS sample buffer and 1× reducing agent prior to boiling for 5 min at 95°C and subsequently analyzed via immunoblotting.

Flow cytometry

mCherry-GFP-APP lysosome delivery reporter:

Flow cytometry analysis of mCherry-GFP-APP-expressing HeLa cells was performed as previously described [48].

ADRB2 receptor recycling assay

HEK293T cells were plated in 6-well plates and the next day co-transfected with cDNAs encoding HA-FLAG-ADRB2 (150 ng), mCherry (300 ng) and empty vector up to 1500 ng using the BES-calcium chloride method. Two days later, cells were pre-treated with or without AZD8055 for 6 h, then endocytosis of the receptor was stimulated with a 30 min isoproterenol “pulse”. To allow internalized receptors to recycle back to the cell surface, cells were washed with warm DPBS and then “chased” with full medium containing Alprenalol for 10 min (the antagonist prevents endocytosis from residual agonist, as described previously [6]). Cells were then washed with cold DPBS, detached in an enzyme-free manner with Versene (0.53 mM EDTA-PBS) at 4°C and fixed with 5% formalin for 15 min. Cells were blocked with 5% normal donkey serum for 30 min, and surface labeled with anti-FLAG antibody diluted in 2% BSA/PBS for 30 min at 4°C. Cells were washed and immuno-stained with Alexa Fluor 488-conjugated secondary antibody diluted in 2% BSA-PBS for 30 min in the dark. Cells were re-washed and resuspended in 2% BSA-PBS prior to flow cytometry analysis (~200,000 events/condition). Data analysis was performed using FlowJo.

In the single cell population, mCherry⁺ signal was used to distinguish transfected cells, and Alexa Fluor488⁺ signal indicated cell surface levels of HA-FLAG-ADRB2 (i.e., because the HA-FLAG-tag is situated on the extracellular N terminus). The number of double-positive cells (mCherry⁺ and Alexa Fluor 488⁺, i.e., transfected cells with HA-FLAG-ADRB2 on their plasma membrane) was divided by the total number of transfected cells (mCherry⁺) to give their frequency. The frequency of double-positive cells in the “chase” condition was subtracted from that in the “pulse” condition to give a “recycling index” which was normalized to the control group. Key parameters of this assay were empirically determined to be within linear range (e.g., cDNA amount and chase time).

Statistical analysis

The statistical analyses performed are explicitly stated in the figure legend. Gaussian (normal) distribution was verified using the Shapiro-Wilk test. Parametric analyses were used for normally distributed data and non-parametric analyses were used for non-normally distributed data. All statistical analyses were performed using Prism 8 software except for proteomic data (stated above). Values report the mean ± SEM unless otherwise stated. Statistical significance was attributed for P < 0.05. Graphs were annotated with asterisk (*) to denote P values as follows: *P < 0.05, **P < 0.01, ***P < 0.001and ****P < 0.0001.

Supplementary Material

Carosi et al Supplemental Figures R4.docx

KAUP_A_2281126_SM5996.docx^{(21.9MB, docx)}

Table S1 R4.xlsx

KAUP_A_2281126_SM5995.xlsx^{(7MB, xlsx)}

Acknowledgements

We thank all laboratory members for insightful discussions and technical advice. We thank Rohan D. Teasdale (University of Queensland) for sharing knock-out cells; Yoon Lim (Centre for Cancer Biology) for assistance with optimising immunoprecipitations; Makoto Kamei (Centre for Cancer Biology & SAHMRI) for assistance with confocal microscopy. Randall Grose and Jarrod Goyne (SAHMRI) for assistance with flow cytometry. Christopher Proud and laboratory (SAHMRI) for sharing plasmids and reagents; Pirjo Apaja (SAHMRI) for sharing reagents. Plasmids from Addgene are indicated in the Materials and Methods section.

This investigation was supported by Lysosomal Health in Ageing at SAHMRI. JMC was supported by a Research Training Stipend and a Commonwealth Scholarship from the Australian Government, a Research Degree Excellence Grant from UniSA, and an EMCR Fellowship from The Hospital Research Foundation Group (2022-CF-EMCR-007). DD was supported by a National Health & Medical Research Council (NHMRC) Project Grant (1124490), an Australian Research Council Discovery Project (DP10100665) and a UniSA Support Package to SK. SK was supported by a NHMRC Senior Principal Research Fellowship (GNT1103006) and a L3 Investigator Grant (2007739).

Funding Statement

The work was supported by the Australian Government Australian Research Council [DP10100665]; National Health and Medical Research Council [GNT1103006]; National Health and Medical Research Council [2007739]; National Health and Medical Research Council [1124490]; University of South Australia; The Hospital Research Foundation Group [2022-CF-EMCR-007].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2281126

References

[1].Carosi JM, Denton D, Kumar S, et al. Receptor recycling by Retromer. Mol Cell Biol. 2023;43(7):317–334. doi: 10.1080/10985549.2023.2222053 [DOI] [PMC free article] [PubMed] [Google Scholar]
[2].Seaman MN. Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J Cell Biol. 2004;165(1):111–122. doi: 10.1083/jcb.200312034 [DOI] [PMC free article] [PubMed] [Google Scholar]
[3].Seaman MN, Marcusson EG, Cereghino JL, et al. Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products. J Cell Biol. 1997;137(1):79–92. doi: 10.1083/jcb.137.1.79 [DOI] [PMC free article] [PubMed] [Google Scholar]
[4].Harbour ME, Breusegem SYA, Antrobus R, et al. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics. J Cell Sci. 2010;123(21):3703–3717. doi: 10.1242/jcs.071472 [DOI] [PMC free article] [PubMed] [Google Scholar]
[5].Hesketh GG, Pérez-Dorado I, Jackson L, et al. VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface. Dev Cell. 2014;29(5):591–606. doi: 10.1016/j.devcel.2014.04.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
[6].Temkin P, Lauffer B, Jäger S, et al. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. Nat Cell Biol. 2011;13(6):715–721. doi: 10.1038/ncb2252 [DOI] [PMC free article] [PubMed] [Google Scholar]
[7].Carlton J, Bujny M, Peter BJ, et al. Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides. Curr Biol. 2004;14(20):1791–1800. doi: 10.1016/j.cub.2004.09.077 [DOI] [PubMed] [Google Scholar]
[8].Rojas R, Kametaka S, Haft CR, et al. Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol Cell Biol. 2007;27(3):1112–1124. doi: 10.1128/MCB.00156-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
[9].Wassmer T, Attar N, Bujny MV, et al. A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer. J Cell Sci. 2007;120(1):45–54. doi: 10.1242/jcs.03302 [DOI] [PubMed] [Google Scholar]
[10].Harterink M, Port F, Lorenowicz MJ, et al. A SNX3-dependent retromer pathway mediates retrograde transport of the wnt sorting receptor wntless and is required for wnt secretion. Nat Cell Biol. 2011;13(8):914–923. doi: 10.1038/ncb2281 [DOI] [PMC free article] [PubMed] [Google Scholar]
[11].Steinberg F, Gallon M, Winfield M, et al. A global analysis of SNX27–retromer assembly and cargo specificity reveals a function in glucose and metal ion transport. Nat Cell Biol. 2013;15(5):461–471. doi: 10.1038/ncb2721 [DOI] [PMC free article] [PubMed] [Google Scholar]
[12].Harrison MS, Hung C-S, Liu T-T, et al. A mechanism for retromer endosomal coat complex assembly with cargo. Proc Natl Acad Sci U S A. 2014;111(1):267–272. doi: 10.1073/pnas.1316482111 [DOI] [PMC free article] [PubMed] [Google Scholar]
[13].Seaman MN, Harbour ME, Tattersall D, et al. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the rab-GAP TBC1D5. J Cell Sci. 2009;122(Pt 14):2371–2382. doi: 10.1242/jcs.048686 [DOI] [PMC free article] [PubMed] [Google Scholar]
[14].Rojas R, van Vlijmen T, Mardones GA, et al. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J Cell Biol. 2008;183(3):513–526. doi: 10.1083/jcb.200804048 [DOI] [PMC free article] [PubMed] [Google Scholar]
[15].Seaman MN. Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval. J Cell Sci. 2007;120(Pt 14):2378–2389. doi: 10.1242/jcs.009654 [DOI] [PubMed] [Google Scholar]
[16].Lucas M, Gershlick DC, Vidaurrazaga A, et al. Structural mechanism for cargo Recognition by the retromer complex. Cell. 2016;167(6):1623–1635. doi: 10.1016/j.cell.2016.10.056 [DOI] [PMC free article] [PubMed] [Google Scholar]
[17].Gomez TS, Billadeau DD. A FAM21-containing WASH complex regulates retromer-dependent sorting. Dev Cell. 2009;17(5):699–711. doi: 10.1016/j.devcel.2009.09.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
[18].Wassmer T, Attar N, Harterink M, et al. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Dev Cell. 2009;17(1):110–122. doi: 10.1016/j.devcel.2009.04.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
[19].Cui Y, Carosi JM, Yang Z, et al. Retromer has a selective function in cargo sorting via endosome transport carriers. J Cell Biol. 2019;218(2):615–631. doi: 10.1083/jcb.201806153 [DOI] [PMC free article] [PubMed] [Google Scholar]
[20].Jia D, Zhang J-S, Li F, et al. Structural and mechanistic insights into regulation of the retromer coat by TBC1d5. Nat Commun. 2016;7(1):1–11. doi: 10.1038/ncomms13305 [DOI] [PMC free article] [PubMed] [Google Scholar]
[21].Borg Distefano M, Hofstad Haugen L, Wang Y, et al. TBC1D5 controls the GTPase cycle of Rab7b. J Cell Sci. 2018;131(17). doi: 10.1242/jcs.216630 [DOI] [PubMed] [Google Scholar]
[22].Kaur J, Debnath J. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 2015;16(8):461–472. doi: 10.1038/nrm4024 [DOI] [PubMed] [Google Scholar]
[23].Kim J, Kundu M, Viollet B, et al. AMPK and MTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13(2):132–141. doi: 10.1038/ncb2152 [DOI] [PMC free article] [PubMed] [Google Scholar]
[24].Carosi JM, Fourrier C, Bensalem J, et al. The mTOR–lysosome axis at the centre of ageing. FEBS Open Bio. 2022;12(4):739–757. doi: 10.1002/2211-5463.13347 [DOI] [PMC free article] [PubMed] [Google Scholar]
[25].Ganley IG, Lam DH, Wang J, et al. ULK1·ATG13·FIP200 complex mediates mTOR Signaling and is essential for autophagy. J Biol Chem. 2009;284(18):12297–12305. doi: 10.1074/jbc.M900573200 [DOI] [PMC free article] [PubMed] [Google Scholar]
[26].Bensalem J, Hattersley, KJ, Hein, LK, et al. Measurement of autophagic flux in humans: an optimized method for blood samples. Autophagy. 2020;16(1):1–18. doi: 10.1080/15548627.2019.1665293 [DOI] [PMC free article] [PubMed] [Google Scholar]
[27].Kvainickas A, Nägele H, Qi W, et al. Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid–induced mTORC1 signaling. J Cell Biol. 2019;218(9):3019–3038. doi: 10.1083/jcb.201812110 [DOI] [PMC free article] [PubMed] [Google Scholar]
[28].Zavodszky E, Seaman MNJ, Moreau K, et al. Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nat Commun. 2014;5(1):1–16. doi: 10.1038/ncomms4828 [DOI] [PMC free article] [PubMed] [Google Scholar]
[29].Jimenez-Orgaz A, Kvainickas A, Nägele H, et al. Control of RAB7 activity and localization through the retromer-TBC1D5 complex enables RAB7-dependent mitophagy. EMBO J. 2018;37(2):235–254. doi: 10.15252/embj.201797128 [DOI] [PMC free article] [PubMed] [Google Scholar]
[30].Carosi JM, Hein LK, van den Hurk M, et al. Retromer regulates the lysosomal clearance of MAPT/tau. Autophagy. 2020;16(1):1–21. doi: 10.1080/15548627.2019.1665293 [DOI] [PMC free article] [PubMed] [Google Scholar]
[31].Carosi JM, Denton D, Kumar S, et al. Retromer dysfunction at the nexus of tauopathies. Cell Death Differ. 2021;28(3):884–899. doi: 10.1038/s41418-020-00727-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
[32].Arighi CN, Hartnell LM, Aguilar RC, et al. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J Cell Biol. 2004;165(1):123–133. doi: 10.1083/jcb.200312055 [DOI] [PMC free article] [PubMed] [Google Scholar]
[33].Carosi JM, Hattersley KJ, Cui Y, et al. Subcellular fractionation of hela cells for lysosome enrichment using a continuous percoll-density gradient. Bio-protocol. 2019;9(18):e3362. doi: 10.21769/BioProtoc.3362 [DOI] [PMC free article] [PubMed] [Google Scholar]
[34].Kirkin V, Rogov VV. A diversity of selective autophagy receptors determines the specificity of the autophagy pathway. Mol Cell. 2019;76(2):268–285. doi: 10.1016/j.molcel.2019.09.005 [DOI] [PubMed] [Google Scholar]
[35].Popovic D, Akutsu M, Novak I, et al. Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. Mol Biol Cell. 2012;32(9):1733–1744. doi: 10.1128/MCB.06717-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
[36].Mao L, Liao C, Qin J, et al. Phosphorylation of SNX27 by MAPK11/14 links cellular stress–signaling pathways with endocytic recycling. J Cell Biol. 2021;220(4). doi: 10.1083/jcb.202010048 [DOI] [PMC free article] [PubMed] [Google Scholar]
[37].Roy S, Leidal AM, Ye J, et al. Autophagy-dependent shuttling of TBC1D5 controls plasma membrane translocation of GLUT1 and glucose uptake. Mol Cell. 2017;67(1):84–95 e5. doi: 10.1016/j.molcel.2017.05.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
[38].Roy S, Debnath J. Autophagy enables retromer-dependent plasma membrane translocation of SLC2A1/GLUT1 to enhance glucose uptake. Autophagy. 2017;13(11):2013–2014. doi: 10.1080/15548627.2017.1371397 [DOI] [PMC free article] [PubMed] [Google Scholar]
[39].Popovic D, Dikic I. TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy. EMBO Rep. 2014;15(4):392–401. doi: 10.1002/embr.201337995 [DOI] [PMC free article] [PubMed] [Google Scholar]
[40].Burnett PE, Barrow RK, Cohen NA, et al. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A. 1998;95(4):1432–1437. doi: 10.1073/pnas.95.4.1432 [DOI] [PMC free article] [PubMed] [Google Scholar]
[41].Kang SA, Pacold ME, Cervantes CL, et al. MTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science. 2013;341(6144):1236566. doi: 10.1126/science.1236566 [DOI] [PMC free article] [PubMed] [Google Scholar]
[42].Huang S, Yang ZJ, Yu C, et al. Inhibition of MTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1. J Biol Chem. 2011;286(46):40002–40012. doi: 10.1074/jbc.M111.297432 [DOI] [PMC free article] [PubMed] [Google Scholar]
[43].Willems L, Chapuis N, Puissant A, et al. The dual MTORC1 and MTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia. Leukemia. 2012;26(6):1195–1202. doi: 10.1038/leu.2011.339 [DOI] [PubMed] [Google Scholar]
[44].Egan DF, Shackelford DB, Mihaylova MM, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011;331(6016):456–461. doi: 10.1126/science.1196371 [DOI] [PMC free article] [PubMed] [Google Scholar]
[45].Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-MTOR complex. Science. 2005;307(5712):1098–1101. doi: 10.1126/science.1106148 [DOI] [PubMed] [Google Scholar]
[46].Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibits MTORC2 assembly and Akt/PKB. Mol Cell. 2006;22(2):159–168. doi: 10.1016/j.molcel.2006.03.029 [DOI] [PubMed] [Google Scholar]
[47].Orozco JM, Krawczyk PA, Scaria SM, et al. Dihydroxyacetone phosphate signals glucose availability to MTORC1. Nat Metab. 2020;2(9):893–901. doi: 10.1038/s42255-020-0250-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
[48].Hein LK, Apaja PM, Hattersley K, et al. A novel fluorescent probe reveals starvation controls the commitment of amyloid precursor protein to the lysosome. Biochim Biophys Acta, Mol Cell Res. 2017;1864(10):1554–1565. doi: 10.1016/j.bbamcr.2017.06.011 [DOI] [PubMed] [Google Scholar]
[49].Xie J, De Poi SP, Humphrey SJ, et al. TSC-insensitive rheb mutations induce oncogenic transformation through a combination of constitutively active MTORC1 signalling and proteome remodelling. Cell Mol Life Sci. 2021;78(8):4035–4052. doi: 10.1007/s00018-021-03825-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
[50].Wang Y, Hong X, Wang J, et al. Inhibition of MAPK pathway is essential for suppressing rheb-Y35N driven tumor growth. Oncogene. 2017;36(6):756–765. doi: 10.1038/onc.2016.246 [DOI] [PubMed] [Google Scholar]
[51].Hanada T, Noda NN, Satomi Y, et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem. 2007;282(52):37298–37302. doi: 10.1074/jbc.C700195200 [DOI] [PubMed] [Google Scholar]
[52].Nguyen TN, Lazarou M. A unifying model for the role of the ATG8 system in autophagy. J Cell Sci. 2022;135(11):135(11. doi: 10.1242/jcs.258997 [DOI] [PubMed] [Google Scholar]
[53].Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3(5):452–460. doi: 10.4161/auto.4451 [DOI] [PubMed] [Google Scholar]
[54].McEwan DG, Popovic D, Gubas A, et al. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol Cell. 2015;57(1):39–54. doi: 10.1016/j.molcel.2014.11.006 [DOI] [PubMed] [Google Scholar]
[55].Zellner S, Schifferer M, Behrends C. Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling. Mol Cell. 2021;81(6):1337–1354.e8. doi: 10.1016/j.molcel.2021.01.009 [DOI] [PubMed] [Google Scholar]
[56].McNally KE, Faulkner R, Steinberg F, et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat Cell Biol. 2017;19(10):1214–1225. doi: 10.1038/ncb3610 [DOI] [PMC free article] [PubMed] [Google Scholar]
[57].Jacomin AC, Samavedam S, Promponas V, et al. iLIR database: a web resource for LIR motif-containing proteins in eukaryotes. Autophagy. 2016;12(10):1945–1953. doi: 10.1080/15548627.2016.1207016 [DOI] [PMC free article] [PubMed] [Google Scholar]
[58].Fjorback AW, Seaman M, Gustafsen C, et al. Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing. J Neurosci. 2012;32(4):1467–1480. doi: 10.1523/JNEUROSCI.2272-11.2012 [DOI] [PMC free article] [PubMed] [Google Scholar]
[59].Bhalla A, Vetanovetz CP, Morel E, et al. The location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport. Neurobiol Dis. 2012;47(1):126–134. doi: 10.1016/j.nbd.2012.03.030 [DOI] [PMC free article] [PubMed] [Google Scholar]
[60].Curnock R, Calcagni A, Ballabio A, et al. TFEB controls retromer expression in response to nutrient availability. J Cell Biol. 2019;218(12):3954–3966. doi: 10.1083/jcb.201903006 [DOI] [PMC free article] [PubMed] [Google Scholar]
[61].Shi M, Chen B, Mahajan D, et al. Amino acids stimulate the endosome-to-Golgi trafficking through ragulator and small GTPase Arl5. Nat Commun. 2018;9(1):4987. doi: 10.1038/s41467-018-07444-y [DOI] [PMC free article] [PubMed] [Google Scholar]
[62].Xie MW, Jin F, Hwang H, et al. Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method. Proc Natl Acad Sci U S A. 2005;102(20):7215–7220. doi: 10.1073/pnas.0500297102 [DOI] [PMC free article] [PubMed] [Google Scholar]
[63].Müller M, Schmidt O, Angelova M, et al. The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation. Elife. 2015;4:e07736. doi: 10.7554/eLife.07736 [DOI] [PMC free article] [PubMed] [Google Scholar]
[64].Yin P, Hong Z, Zhang L, et al. Retromer localizes to autophagosomes during HCV replication. Virol Sin. 2017;32(3):245–248. doi: 10.1007/s12250-016-3914-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
[65].Dengjel J, Høyer-Hansen M, Nielsen MO, et al. Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens. Mol Cell Proteomics. 2012;11(3):M111.014035. doi: 10.1074/mcp.M111.014035 [DOI] [PMC free article] [PubMed] [Google Scholar]
[66].Mancias JD, Wang X, Gygi SP, et al. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature. 2014;509(7498):105–109. doi: 10.1038/nature13148 [DOI] [PMC free article] [PubMed] [Google Scholar]
[67].Muscolino E, Schmitz R, Loroch S, et al. Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism. Nat Microbiol. 2020;5(2):331–342. doi: 10.1038/s41564-019-0624-1 [DOI] [PubMed] [Google Scholar]
[68].Zhou C, Wu Z, Du W, et al. Recycling of autophagosomal components from autolysosomes by the recycler complex. Nat Cell Biol. 2022;24(4):497–512. doi: 10.1038/s41556-022-00861-8 [DOI] [PubMed] [Google Scholar]
[69].Seaman MNJ, Mukadam AS, Breusegem SY. Inhibition of TBC1D5 activates Rab7a and can enhance the function of the retromer cargo-selective complex. J Cell Sci. 2018;131(12). doi: 10.1242/jcs.217398 [DOI] [PMC free article] [PubMed] [Google Scholar]
[70].Huttlin EL, Bruckner RJ, Navarrete-Perea J, et al. Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell. 2021;184(11):3022–3040.e28. doi: 10.1016/j.cell.2021.04.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
[71].du Toit A, Hofmeyr J-HS, Gniadek TJ, et al. Measuring autophagosome flux. Autophagy. 2018;14(6):1060–1071. doi: 10.1080/15548627.2018.1469590 [DOI] [PMC free article] [PubMed] [Google Scholar]
[72].Crawley-Snowdon H, Yang J-C, Zaccai NR, et al. Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29. Nat Commun. 2020;11(1):5031. doi: 10.1038/s41467-020-18773-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
[73].Hassiotis S, Manavis J, Blumbergs PC, et al. Lysosomal LAMP1 immunoreactivity exists in both diffuse and neuritic amyloid plaques in the human hippocampus. Eur J Neurosci. 2018;47(9):1043–1053. doi: 10.1111/ejn.13913 [DOI] [PubMed] [Google Scholar]
[74].Tang Y, Hu LA, Miller WE, et al. Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor. Proc Natl Acad Sci USA. 1999;96(22):12559–12564. doi: 10.1073/pnas.96.22.12559 [DOI] [PMC free article] [PubMed] [Google Scholar]
[75].Rowland AA, Chitwood PJ, Phillips MJ, et al. ER contact sites define the position and timing of endosome fission. Cell. 2014;159(5):1027–1041. doi: 10.1016/j.cell.2014.10.023 [DOI] [PMC free article] [PubMed] [Google Scholar]
[76].Hazelbaker DZ, Beccard A, Bara AM, et al. A scaled framework for CRISPR editing of human pluripotent stem cells to study psychiatric disease. Stem Cell Rep. 2017;9(4):1315–1327. doi: 10.1016/j.stemcr.2017.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
[77].Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11(8):783–784. doi: 10.1038/nmeth.3047 [DOI] [PMC free article] [PubMed] [Google Scholar]
[78].Stewart SA, Dykxhoorn DM, Palliser D, et al. C. RNA. 2003;9(4):493–501. doi: 10.1261/rna.2192803 [DOI] [PMC free article] [PubMed] [Google Scholar]
[79].Pettersen EF, Goddard TD, Huang CC, et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 2021;30(1):70–82. doi: 10.1002/pro.3943 [DOI] [PMC free article] [PubMed] [Google Scholar]
[80].Xue B, Dunbrack RL, Williams RW, et al. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim Biophys Acta. 2010;1804(4):996–1010. doi: 10.1016/j.bbapap.2010.01.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
[81].Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–589. doi: 10.1038/s41586-021-03819-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
[82].Kingston RE, Chen CA, Rose JK. Calcium phosphate transfection. Curr Protoc Mol Biol. 2003;63(1):9–1. doi: 10.1002/0471142727.mb0901s63 [DOI] [PubMed] [Google Scholar]
[83].Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006;224(Pt 3):213–232. doi: 10.1111/j.1365-2818.2006.01706.x [DOI] [PubMed] [Google Scholar]
[84].Wiśniewski JR, Zougman A, Nagaraj N, et al. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6(5):359–362. doi: 10.1038/nmeth.1322 [DOI] [PubMed] [Google Scholar]
[85].Nguyen TN, Padman BS, Usher J, et al. Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J Cell Biol. 2016;215(6):857–874. doi: 10.1083/jcb.201607039 [DOI] [PMC free article] [PubMed] [Google Scholar]

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Table S1 R4.xlsx

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[cit0001] [1].Carosi JM, Denton D, Kumar S, et al. Receptor recycling by Retromer. Mol Cell Biol. 2023;43(7):317–334. doi: 10.1080/10985549.2023.2222053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0002] [2].Seaman MN. Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J Cell Biol. 2004;165(1):111–122. doi: 10.1083/jcb.200312034 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0003] [3].Seaman MN, Marcusson EG, Cereghino JL, et al. Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products. J Cell Biol. 1997;137(1):79–92. doi: 10.1083/jcb.137.1.79 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0004] [4].Harbour ME, Breusegem SYA, Antrobus R, et al. The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics. J Cell Sci. 2010;123(21):3703–3717. doi: 10.1242/jcs.071472 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0005] [5].Hesketh GG, Pérez-Dorado I, Jackson L, et al. VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface. Dev Cell. 2014;29(5):591–606. doi: 10.1016/j.devcel.2014.04.010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0006] [6].Temkin P, Lauffer B, Jäger S, et al. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. Nat Cell Biol. 2011;13(6):715–721. doi: 10.1038/ncb2252 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0007] [7].Carlton J, Bujny M, Peter BJ, et al. Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides. Curr Biol. 2004;14(20):1791–1800. doi: 10.1016/j.cub.2004.09.077 [DOI] [PubMed] [Google Scholar]

[cit0008] [8].Rojas R, Kametaka S, Haft CR, et al. Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors. Mol Cell Biol. 2007;27(3):1112–1124. doi: 10.1128/MCB.00156-06 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0009] [9].Wassmer T, Attar N, Bujny MV, et al. A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer. J Cell Sci. 2007;120(1):45–54. doi: 10.1242/jcs.03302 [DOI] [PubMed] [Google Scholar]

[cit0010] [10].Harterink M, Port F, Lorenowicz MJ, et al. A SNX3-dependent retromer pathway mediates retrograde transport of the wnt sorting receptor wntless and is required for wnt secretion. Nat Cell Biol. 2011;13(8):914–923. doi: 10.1038/ncb2281 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0011] [11].Steinberg F, Gallon M, Winfield M, et al. A global analysis of SNX27–retromer assembly and cargo specificity reveals a function in glucose and metal ion transport. Nat Cell Biol. 2013;15(5):461–471. doi: 10.1038/ncb2721 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0012] [12].Harrison MS, Hung C-S, Liu T-T, et al. A mechanism for retromer endosomal coat complex assembly with cargo. Proc Natl Acad Sci U S A. 2014;111(1):267–272. doi: 10.1073/pnas.1316482111 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0013] [13].Seaman MN, Harbour ME, Tattersall D, et al. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the rab-GAP TBC1D5. J Cell Sci. 2009;122(Pt 14):2371–2382. doi: 10.1242/jcs.048686 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0014] [14].Rojas R, van Vlijmen T, Mardones GA, et al. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7. J Cell Biol. 2008;183(3):513–526. doi: 10.1083/jcb.200804048 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0015] [15].Seaman MN. Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval. J Cell Sci. 2007;120(Pt 14):2378–2389. doi: 10.1242/jcs.009654 [DOI] [PubMed] [Google Scholar]

[cit0016] [16].Lucas M, Gershlick DC, Vidaurrazaga A, et al. Structural mechanism for cargo Recognition by the retromer complex. Cell. 2016;167(6):1623–1635. doi: 10.1016/j.cell.2016.10.056 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0017] [17].Gomez TS, Billadeau DD. A FAM21-containing WASH complex regulates retromer-dependent sorting. Dev Cell. 2009;17(5):699–711. doi: 10.1016/j.devcel.2009.09.009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0018] [18].Wassmer T, Attar N, Harterink M, et al. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Dev Cell. 2009;17(1):110–122. doi: 10.1016/j.devcel.2009.04.016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0019] [19].Cui Y, Carosi JM, Yang Z, et al. Retromer has a selective function in cargo sorting via endosome transport carriers. J Cell Biol. 2019;218(2):615–631. doi: 10.1083/jcb.201806153 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0020] [20].Jia D, Zhang J-S, Li F, et al. Structural and mechanistic insights into regulation of the retromer coat by TBC1d5. Nat Commun. 2016;7(1):1–11. doi: 10.1038/ncomms13305 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0021] [21].Borg Distefano M, Hofstad Haugen L, Wang Y, et al. TBC1D5 controls the GTPase cycle of Rab7b. J Cell Sci. 2018;131(17). doi: 10.1242/jcs.216630 [DOI] [PubMed] [Google Scholar]

[cit0022] [22].Kaur J, Debnath J. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 2015;16(8):461–472. doi: 10.1038/nrm4024 [DOI] [PubMed] [Google Scholar]

[cit0023] [23].Kim J, Kundu M, Viollet B, et al. AMPK and MTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13(2):132–141. doi: 10.1038/ncb2152 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0024] [24].Carosi JM, Fourrier C, Bensalem J, et al. The mTOR–lysosome axis at the centre of ageing. FEBS Open Bio. 2022;12(4):739–757. doi: 10.1002/2211-5463.13347 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0025] [25].Ganley IG, Lam DH, Wang J, et al. ULK1·ATG13·FIP200 complex mediates mTOR Signaling and is essential for autophagy. J Biol Chem. 2009;284(18):12297–12305. doi: 10.1074/jbc.M900573200 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0026] [26].Bensalem J, Hattersley, KJ, Hein, LK, et al. Measurement of autophagic flux in humans: an optimized method for blood samples. Autophagy. 2020;16(1):1–18. doi: 10.1080/15548627.2019.1665293 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0027] [27].Kvainickas A, Nägele H, Qi W, et al. Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid–induced mTORC1 signaling. J Cell Biol. 2019;218(9):3019–3038. doi: 10.1083/jcb.201812110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0028] [28].Zavodszky E, Seaman MNJ, Moreau K, et al. Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nat Commun. 2014;5(1):1–16. doi: 10.1038/ncomms4828 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0029] [29].Jimenez-Orgaz A, Kvainickas A, Nägele H, et al. Control of RAB7 activity and localization through the retromer-TBC1D5 complex enables RAB7-dependent mitophagy. EMBO J. 2018;37(2):235–254. doi: 10.15252/embj.201797128 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0030] [30].Carosi JM, Hein LK, van den Hurk M, et al. Retromer regulates the lysosomal clearance of MAPT/tau. Autophagy. 2020;16(1):1–21. doi: 10.1080/15548627.2019.1665293 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0031] [31].Carosi JM, Denton D, Kumar S, et al. Retromer dysfunction at the nexus of tauopathies. Cell Death Differ. 2021;28(3):884–899. doi: 10.1038/s41418-020-00727-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0032] [32].Arighi CN, Hartnell LM, Aguilar RC, et al. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J Cell Biol. 2004;165(1):123–133. doi: 10.1083/jcb.200312055 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0033] [33].Carosi JM, Hattersley KJ, Cui Y, et al. Subcellular fractionation of hela cells for lysosome enrichment using a continuous percoll-density gradient. Bio-protocol. 2019;9(18):e3362. doi: 10.21769/BioProtoc.3362 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0034] [34].Kirkin V, Rogov VV. A diversity of selective autophagy receptors determines the specificity of the autophagy pathway. Mol Cell. 2019;76(2):268–285. doi: 10.1016/j.molcel.2019.09.005 [DOI] [PubMed] [Google Scholar]

[cit0035] [35].Popovic D, Akutsu M, Novak I, et al. Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. Mol Biol Cell. 2012;32(9):1733–1744. doi: 10.1128/MCB.06717-11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0036] [36].Mao L, Liao C, Qin J, et al. Phosphorylation of SNX27 by MAPK11/14 links cellular stress–signaling pathways with endocytic recycling. J Cell Biol. 2021;220(4). doi: 10.1083/jcb.202010048 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0037] [37].Roy S, Leidal AM, Ye J, et al. Autophagy-dependent shuttling of TBC1D5 controls plasma membrane translocation of GLUT1 and glucose uptake. Mol Cell. 2017;67(1):84–95 e5. doi: 10.1016/j.molcel.2017.05.020 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0038] [38].Roy S, Debnath J. Autophagy enables retromer-dependent plasma membrane translocation of SLC2A1/GLUT1 to enhance glucose uptake. Autophagy. 2017;13(11):2013–2014. doi: 10.1080/15548627.2017.1371397 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0039] [39].Popovic D, Dikic I. TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy. EMBO Rep. 2014;15(4):392–401. doi: 10.1002/embr.201337995 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0040] [40].Burnett PE, Barrow RK, Cohen NA, et al. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci U S A. 1998;95(4):1432–1437. doi: 10.1073/pnas.95.4.1432 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0041] [41].Kang SA, Pacold ME, Cervantes CL, et al. MTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science. 2013;341(6144):1236566. doi: 10.1126/science.1236566 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0042] [42].Huang S, Yang ZJ, Yu C, et al. Inhibition of MTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1. J Biol Chem. 2011;286(46):40002–40012. doi: 10.1074/jbc.M111.297432 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0043] [43].Willems L, Chapuis N, Puissant A, et al. The dual MTORC1 and MTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia. Leukemia. 2012;26(6):1195–1202. doi: 10.1038/leu.2011.339 [DOI] [PubMed] [Google Scholar]

[cit0044] [44].Egan DF, Shackelford DB, Mihaylova MM, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011;331(6016):456–461. doi: 10.1126/science.1196371 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0045] [45].Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-MTOR complex. Science. 2005;307(5712):1098–1101. doi: 10.1126/science.1106148 [DOI] [PubMed] [Google Scholar]

[cit0046] [46].Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibits MTORC2 assembly and Akt/PKB. Mol Cell. 2006;22(2):159–168. doi: 10.1016/j.molcel.2006.03.029 [DOI] [PubMed] [Google Scholar]

[cit0047] [47].Orozco JM, Krawczyk PA, Scaria SM, et al. Dihydroxyacetone phosphate signals glucose availability to MTORC1. Nat Metab. 2020;2(9):893–901. doi: 10.1038/s42255-020-0250-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0048] [48].Hein LK, Apaja PM, Hattersley K, et al. A novel fluorescent probe reveals starvation controls the commitment of amyloid precursor protein to the lysosome. Biochim Biophys Acta, Mol Cell Res. 2017;1864(10):1554–1565. doi: 10.1016/j.bbamcr.2017.06.011 [DOI] [PubMed] [Google Scholar]

[cit0049] [49].Xie J, De Poi SP, Humphrey SJ, et al. TSC-insensitive rheb mutations induce oncogenic transformation through a combination of constitutively active MTORC1 signalling and proteome remodelling. Cell Mol Life Sci. 2021;78(8):4035–4052. doi: 10.1007/s00018-021-03825-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0050] [50].Wang Y, Hong X, Wang J, et al. Inhibition of MAPK pathway is essential for suppressing rheb-Y35N driven tumor growth. Oncogene. 2017;36(6):756–765. doi: 10.1038/onc.2016.246 [DOI] [PubMed] [Google Scholar]

[cit0051] [51].Hanada T, Noda NN, Satomi Y, et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem. 2007;282(52):37298–37302. doi: 10.1074/jbc.C700195200 [DOI] [PubMed] [Google Scholar]

[cit0052] [52].Nguyen TN, Lazarou M. A unifying model for the role of the ATG8 system in autophagy. J Cell Sci. 2022;135(11):135(11. doi: 10.1242/jcs.258997 [DOI] [PubMed] [Google Scholar]

[cit0053] [53].Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3(5):452–460. doi: 10.4161/auto.4451 [DOI] [PubMed] [Google Scholar]

[cit0054] [54].McEwan DG, Popovic D, Gubas A, et al. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol Cell. 2015;57(1):39–54. doi: 10.1016/j.molcel.2014.11.006 [DOI] [PubMed] [Google Scholar]

[cit0055] [55].Zellner S, Schifferer M, Behrends C. Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling. Mol Cell. 2021;81(6):1337–1354.e8. doi: 10.1016/j.molcel.2021.01.009 [DOI] [PubMed] [Google Scholar]

[cit0056] [56].McNally KE, Faulkner R, Steinberg F, et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat Cell Biol. 2017;19(10):1214–1225. doi: 10.1038/ncb3610 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0057] [57].Jacomin AC, Samavedam S, Promponas V, et al. iLIR database: a web resource for LIR motif-containing proteins in eukaryotes. Autophagy. 2016;12(10):1945–1953. doi: 10.1080/15548627.2016.1207016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0058] [58].Fjorback AW, Seaman M, Gustafsen C, et al. Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing. J Neurosci. 2012;32(4):1467–1480. doi: 10.1523/JNEUROSCI.2272-11.2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0059] [59].Bhalla A, Vetanovetz CP, Morel E, et al. The location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport. Neurobiol Dis. 2012;47(1):126–134. doi: 10.1016/j.nbd.2012.03.030 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0060] [60].Curnock R, Calcagni A, Ballabio A, et al. TFEB controls retromer expression in response to nutrient availability. J Cell Biol. 2019;218(12):3954–3966. doi: 10.1083/jcb.201903006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0061] [61].Shi M, Chen B, Mahajan D, et al. Amino acids stimulate the endosome-to-Golgi trafficking through ragulator and small GTPase Arl5. Nat Commun. 2018;9(1):4987. doi: 10.1038/s41467-018-07444-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0062] [62].Xie MW, Jin F, Hwang H, et al. Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method. Proc Natl Acad Sci U S A. 2005;102(20):7215–7220. doi: 10.1073/pnas.0500297102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0063] [63].Müller M, Schmidt O, Angelova M, et al. The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation. Elife. 2015;4:e07736. doi: 10.7554/eLife.07736 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0064] [64].Yin P, Hong Z, Zhang L, et al. Retromer localizes to autophagosomes during HCV replication. Virol Sin. 2017;32(3):245–248. doi: 10.1007/s12250-016-3914-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0065] [65].Dengjel J, Høyer-Hansen M, Nielsen MO, et al. Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens. Mol Cell Proteomics. 2012;11(3):M111.014035. doi: 10.1074/mcp.M111.014035 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0066] [66].Mancias JD, Wang X, Gygi SP, et al. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature. 2014;509(7498):105–109. doi: 10.1038/nature13148 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0067] [67].Muscolino E, Schmitz R, Loroch S, et al. Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism. Nat Microbiol. 2020;5(2):331–342. doi: 10.1038/s41564-019-0624-1 [DOI] [PubMed] [Google Scholar]

[cit0068] [68].Zhou C, Wu Z, Du W, et al. Recycling of autophagosomal components from autolysosomes by the recycler complex. Nat Cell Biol. 2022;24(4):497–512. doi: 10.1038/s41556-022-00861-8 [DOI] [PubMed] [Google Scholar]

[cit0069] [69].Seaman MNJ, Mukadam AS, Breusegem SY. Inhibition of TBC1D5 activates Rab7a and can enhance the function of the retromer cargo-selective complex. J Cell Sci. 2018;131(12). doi: 10.1242/jcs.217398 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0070] [70].Huttlin EL, Bruckner RJ, Navarrete-Perea J, et al. Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell. 2021;184(11):3022–3040.e28. doi: 10.1016/j.cell.2021.04.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0071] [71].du Toit A, Hofmeyr J-HS, Gniadek TJ, et al. Measuring autophagosome flux. Autophagy. 2018;14(6):1060–1071. doi: 10.1080/15548627.2018.1469590 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0072] [72].Crawley-Snowdon H, Yang J-C, Zaccai NR, et al. Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29. Nat Commun. 2020;11(1):5031. doi: 10.1038/s41467-020-18773-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0073] [73].Hassiotis S, Manavis J, Blumbergs PC, et al. Lysosomal LAMP1 immunoreactivity exists in both diffuse and neuritic amyloid plaques in the human hippocampus. Eur J Neurosci. 2018;47(9):1043–1053. doi: 10.1111/ejn.13913 [DOI] [PubMed] [Google Scholar]

[cit0074] [74].Tang Y, Hu LA, Miller WE, et al. Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor. Proc Natl Acad Sci USA. 1999;96(22):12559–12564. doi: 10.1073/pnas.96.22.12559 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0075] [75].Rowland AA, Chitwood PJ, Phillips MJ, et al. ER contact sites define the position and timing of endosome fission. Cell. 2014;159(5):1027–1041. doi: 10.1016/j.cell.2014.10.023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0076] [76].Hazelbaker DZ, Beccard A, Bara AM, et al. A scaled framework for CRISPR editing of human pluripotent stem cells to study psychiatric disease. Stem Cell Rep. 2017;9(4):1315–1327. doi: 10.1016/j.stemcr.2017.09.006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0077] [77].Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11(8):783–784. doi: 10.1038/nmeth.3047 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0078] [78].Stewart SA, Dykxhoorn DM, Palliser D, et al. C. RNA. 2003;9(4):493–501. doi: 10.1261/rna.2192803 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0079] [79].Pettersen EF, Goddard TD, Huang CC, et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 2021;30(1):70–82. doi: 10.1002/pro.3943 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0080] [80].Xue B, Dunbrack RL, Williams RW, et al. PONDR-FIT: a meta-predictor of intrinsically disordered amino acids. Biochim Biophys Acta. 2010;1804(4):996–1010. doi: 10.1016/j.bbapap.2010.01.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0081] [81].Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–589. doi: 10.1038/s41586-021-03819-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0082] [82].Kingston RE, Chen CA, Rose JK. Calcium phosphate transfection. Curr Protoc Mol Biol. 2003;63(1):9–1. doi: 10.1002/0471142727.mb0901s63 [DOI] [PubMed] [Google Scholar]

[cit0083] [83].Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006;224(Pt 3):213–232. doi: 10.1111/j.1365-2818.2006.01706.x [DOI] [PubMed] [Google Scholar]

[cit0084] [84].Wiśniewski JR, Zougman A, Nagaraj N, et al. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6(5):359–362. doi: 10.1038/nmeth.1322 [DOI] [PubMed] [Google Scholar]

[cit0085] [85].Nguyen TN, Padman BS, Usher J, et al. Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J Cell Biol. 2016;215(6):857–874. doi: 10.1083/jcb.201607039 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling

Julian M Carosi

Leanne K Hein

Jarrod J Sandow

Linh V P Dang

Kathryn Hattersley

Donna Denton

Sharad Kumar

Timothy J Sargeant

ABSTRACT

Introduction

Results

Autophagy inhibition by MTORC1 controls the abundance of retromer+ endosomes

Figure 1.

The retromer-TBC1D5 interaction is not nutrient sensitive

Figure 2.

MTORC1 prevents capture and turnover of retromer-TBC1D5 decorated endosomes by autophagosomes

Figure 3.

The TBC1D5 interaction is critical for retromer to be trafficked to autophagosomes following MTOR inhibition

Figure 4.

Retromer does not interact with RAB7A

MTORC1 supports the trafficking of retromer cargoes

Figure 5.

Discussion

Materials and methods

Table 1.

Molecular cloning

Cell culture

CRISPR-Cas9 gene editing

Transfection

Drug treatments

Starvation experiments

Immunofluorescence, confocal microscopy, and quantification

Analysis of puncta per cell:

Colocalization analysis:

Cell lysis and immunoprecipitation

Immunoblotting

Relative label-free quantitation proteomics

Protease protection assay

Light-membrane and cytoplasm fractionation

Flow cytometry

mCherry-GFP-APP lysosome delivery reporter:

ADRB2 receptor recycling assay

Statistical analysis

Supplementary Material

Acknowledgements

Funding Statement

Disclosure statement

Supplementary material

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Autophagy inhibition by MTORC1 controls the abundance of retromer⁺ endosomes