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Published in final edited form as: Traffic. 2015 Feb 20;16(4):365–378. doi: 10.1111/tra.12253

Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization

Verónica A Segarra 1,*, Douglas R Boettner 1, Sandra K Lemmon 1
PMCID: PMC5599220  NIHMSID: NIHMS903659  PMID: 25557545

Abstract

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the pre-autophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.

Keywords: Autophagy, Cytoplasm to vacuole transport (Cvt), Atg27, Atg9, Tyrosine sorting motif, AP-3, Vacuole, Endosome, Pre-autophagosomal structure (PAS), Trans-Golgi network (TGN)

INTRODUCTION

Autophagy refers to a variety of catabolic processes that target cytoplasmic materials for degradation in the lysosomal/vacuolar lumen. While autophagic processes vary in their specificity for the cytoplasmic components they target for turnover, they are similar in the way in which they sequester and deliver these materials to the lysosome/vacuole (1–3). The unique and distinguishing feature of autophagy is formation of specialized double-membrane transport vesicles known as autophagosomes, which are generated at the pre-autophagosomal structure (PAS). This process and the factors mediating it are highly conserved from yeast to man. Though the membrane trafficking events underlying autophagy and the source of membrane for autophagosome formation are of great interest, they are still not completely understood (1, 4).

Particular attention has been paid to two transmembrane proteins involved in autophagosome formation, Atg9 and, additionally in yeast, Atg27 (5–8). In yeast Atg9 has been found to localize to the PAS and to structures known as Atg9 vesicles, highly mobile single-membrane cytoplasmic vesicles that bud off of the TGN and are funneled to the PAS to aid in early stages of autophagosome formation (5, 8). Additional Atg9 localizes to less-mobile structures, which likely correspond to Atg9 reservoirs in the TGN and endosomal compartments (5, 8–11), that can be mobilized for autophagy. TGN/Endosomal localization of Atg9 in animal cells is also observed and has been suggested to play a similar role (4, 12–14).

Previous work has shown that Atg27 localizes to the PAS and other peripheral structures including the Golgi (6, 15, 16). It is required for proper Atg9 localization to the PAS (5, 15, 16), suggesting it plays a role in anterograde transport of Atg9 to the forming autophagosome. Supporting this, purified Atg9 vesicles also contain Atg27 (17). Furthermore, in studies examining biogenesis of Atg9 vesicles in cells devoid of PAS structures (5), deletion of ATG27 led to Atg9 transport from the TGN to the multivesicular body (MVB)/late endsome and mislocalization to the vacuole lumen (5). It was suggested that this is due to an inability to recruit Atg9 to the TGN-derived transport vesicles destined for the PAS, resulting in Atg9 missorting. However, since Atg9 has been observed in endosomal compartments (10, 11) and Atg27 and Atg9 partially co-localize in structures other than the PAS (6, 15, 16), it is possible that Atg27 plays additional roles in Atg9 trafficking.

To gain further insight into these questions, we have investigated Atg27 localization and trafficking, and its role in Atg9 transport. We uncovered previously undetected endosomal and vacuolar membrane localizations for Atg27. We also report that the vacuolar membrane localization of Atg27 is dependent on both a tyrosine-sorting motif and the heterotetrameric adaptor AP-3. Furthermore, though the Atg27 Y-sorting motif is not required for anterograde transport of Atg9 or Atg27 to the PAS, it is critical for Atg9 retention/retrieval in the TGN/early endosomal compartments.

RESULTS

Atg27 localizes to the vacuolar membrane via the AP-3 pathway

To examine Atg27’s localization and trafficking the coding sequence for GFP was inserted at the 3′ end of the ATG27 open reading frame (18). In the tagging process, we included nucleotides encoding a flexible five-residue glycine-alanine (Gly-Ala) linker between Atg27 and the tag (Figure 1A). The fluorescently-tagged Atg27 reporter is functional (see Supplemental Figure S1). Atg27-GFP localized to a variety of cytoplasmic puncta, which were previously shown to partially colocalize with markers for the PAS, Atg9-positive structures, and the Golgi (8, 15). Although some earlier work suggested that Atg27 localized to mitochondria (15), we did not find Atg27 to be on this organelle (data not shown). To our surprise, we found significant targeting of Atg27 to the vacuolar membrane, which was visualized by FM4-64 staining (Figure 1B). Interestingly, localization of Atg27 to the vacuolar membrane was defective when the Gly-Ala linker between the Atg27 and the GFP tag was absent (Atg27(ΔL)-GFP) (Figure 1C, Supplemental Figure S2). These results may explain why prior studies did not visualize Atg27 at the vacuolar membrane (15, 16) and also suggested the presence of a vacuolar sorting signal in the cytosolic C-terminus of the protein.

Figure 1. Atg27 localization.

Figure 1

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(A) Domains of Atg27 and GFP fusion constructs. SP= signal peptide; TM= transmembrane domain. The red line denotes a five-residue Gly-Ala linker between Atg27 and the GFP tag. (B) Atg27-GFP localizes to the vacuolar membrane and cytoplasmic puncta (SL5837). (C) Atg27(ΔL)-GFP, which lacks the Gly-Ala linker, does not localize to the vacuolar membrane (SL6000). Cells in B and C were allowed to internalize FM4-64 for 30 min to label the vacuole membrane before imaging. The Atg27(ΔL)-GFP construct can be seen at puncta located at the vertices of vacuolar compartments. This was not considered vacuolar membrane localization, as the rim of the vacuole was not marked. These puncta are likely late endosomal compartments, which are, in general, found adjacent to the vacuole (See Figure 4). Scale bar = 5 microns.

We also examined Atg27-GFP localization upon induction of autophagy with rapamycin (Supplemental Figure S2). We found nearly all cells had Atg27-GFP at the vacuolar membrane similar to log phase cells, indicating that localization to the vacuole is not prevented during autophagy.

To determine how Atg27 arrives at the vacuole membrane we tested the effects of blocking major pathways to this lysosome-like compartment. The trafficking itinerary of some membrane proteins involves their cycling through the plasma membrane, which is followed by endocytosis, transport to endosomes and vacuole delivery or recycling to the TGN. To examine whether Atg27 cycles through the plasma membrane, we treated cells with latrunculin A (LAT-A), which prevents assembly of F-actin and inhibits endocytosis (19). Under these conditions, Snc1, a secretory vesicle v-SNARE, accumulates at the cell surface and cannot be recycled (Figure 2A) (20). In contrast, Atg27 did not accumulate at the plasma membrane (Figure 2A), indicating that it does not traffic through the cell surface on its way to the vacuole.

Figure 2. Atg27 vacuolar membrane localization is dependent on the AP-3 pathway.

Figure 2

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(A) Localization of Atg27-GFP and mCherry-Snc1 in cells (SL6498) treated with LAT-A for 2 hours to block endocytosis. (B) Localization of Atg27-mCherry and Cps1-GFP in WT cells (SL6642) and vps4Δ cells (SL6643). (C) Localization of Atg27-GFP in WT cells (SL5837) and apm3Δ cells (SL6213). FM4-64 was used to mark the vacuolar membrane. Scale bar = 5 microns.

In budding yeast, there are two major internal routes from the TGN to the vacuole (21). Cargoes such as the vacuolar hydrolase carboxypeptidase Y (CPY) travel through the prevacuolar compartment (PVC), also known as the multivesicular body (MVB) or late endosome (LE), on their way from the TGN to the vacuole. The second pathway is dependent on the heterotetrameric adaptor AP-3, which mediates transport of cargos directly from the TGN to the vacuole (22, 23). The vacuolar membrane protein alkaline phosphatase (ALP) uses the AP-3 pathway.

To determine if Atg27 arrives at the vacuolar membrane via the PVC/CPY pathway we examined its localization in a vps4Δ mutant. This mutation causes formation of aberrant late endosomes known as class E compartments, from which exit is impaired. Therefore class E endosomes accumulate CPY pathway cargo destined for the vacuole or for recycling to the TGN, as well as cargo entering the LE/MVB from the early endosome (21, 24). When Atg27-mCherry and the CPY pathway cargo GFP-Cps1 were co-expressed in a vps4Δ strain, both accumulated in the class E compartment (Figure 2B), showing that Atg27 traffics through the LE/MVB. However, Atg27’s ability to reach the vacuolar membrane was unaffected, while Cps1 delivery to the vacuolar lumen was blocked.

In contrast, Atg27 was unable reach the vacuolar membrane in an AP-3 μ-chain mutant, apm3Δ (Figure 2C). Deletion of the genes for the μ subunits of the other clathrin AP adaptors, AP-1 and AP-2 (apm1Δ, apm2Δ, apm4Δ) did not alter Atg27’s ability to localize to the vacuolar membrane (Supplemental Figure S3). Overall these data demonstrate that Atg27 traffics both through the endosomal pathway and is an AP-3 dependent cargo.

Atg27 contains a tyrosine sorting motif (YSAV) at its C-terminus

Upon inspection of Atg27’s cytoplasmic C-terminal tail, we noticed that the last four amino acids of the protein (YSAV) conform to the consensus sequence of a classic tyrosine sorting motif YXXΦ, where Y is the key tyrosine residue, X is any amino acid, and Φ is a bulky/hydrophobic amino acid (Figure 3A) (25). Tyrosine sorting motifs can be recognized by the μ chains of heterotetrameric AP adaptor complexes, including AP-3. Atg27 vacuolar membrane localization was greatly impaired upon deletion of the YSAV (Atg27(ΔYSAV)-GFP) or upon mutation of the key tyrosine residue to an alanine (Atg27(ASAV)-GFP) (Figure 3C,D). Atg27 was detected at the vacuolar membrane in less than 15% of cells in the YSAV mutants as compared to nearly 100% in WT cells (Supplemental Figure S2). Therefore, the last four amino acids of Atg27 comprise a tyrosine sorting motif that directs the protein to the vacuolar membrane.

Figure 3. Atg27 localization to the vacuolar membrane is dependent on a tyrosine sorting motif.

Figure 3

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(A) Domains of Atg27 including the C-terminal tyrosine sorting motif (YSAV) and constructs analyzed in B-D. (B-D) Cells were stained with FM4-64 to mark the vacuolar membrane. Localization of (B) WT Atg27-GFP (SL5837), (C) Atg27(ΔYSAV)-GFP (SL5845), and (D) Atg27(ASAV)-GFP (SL6393). Scale bar = 5 microns.

The tyrosine sorting motif affects Atg27 localization in the TGN/endosomal system

We next examined the localization of Atg27 with and without the YSAV to determine how the sorting signal affects colocalization with markers of the TGN/endosomal system. Atg27-GFP displayed partial colocalization with the late Golgi marker Sec7 (Figure 4A) (26, 27). The percentage of Sec7 structures containing Atg27(ΔYSAV)-GFP was approximately doubled relative to cells with wild type (WT) Atg27 (p≤0.001), indicating that the tyrosine sorting motif is important for export from the TGN (Figure 4A). Atg27-GFP and Atg27(ΔYSAV)-GFP each showed partial colocalization with the early and late endosomal markers, Tlg1 and Vps27, respectively (28–31) (Figure 4B, C). However, a higher percentage of early endosomal Tlg1 patches contained Atg27(ΔYSAV) than contained WT Atg27 (86% vs. 51%, respectively, p≤0.01, Figure 4C), while Atg27 and Atg27(ΔYSAV) appeared in Vps27 structures with same frequency (both ~50%) despite Vps27-GFP appearing more cytoplasmic in the wildtype. Overall, these findings suggest that the tyrosine sorting motif mutation affects the distribution of Atg27 in the TGN/endosomal compartments, in addition to its role in sorting Atg27 to the vacuole membrane via the AP-3 pathway.

Figure 4. Atg27 localizes to multiple compartments in the TGN/endosomal system.

Figure 4

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Atg27-XFP and Atg27(ΔYSAV)-XFP (respectively) were localized with the indicated TGN and endosomal markers. (A) Sec7-DsRed, TGN (SL6297 and SL6443); (B) mCherry-Tlg1, early endosomes (SL6495 and SL6500); and (C) Vps27-GFP, late endosome/multivesicular body (SL6604 and SL6605). Bar graphs indicate the % of indicated organelle structures containing Atg27-XFP or Atg27(ΔYSAV)-XFP (n ≥ 200, combined from 3 independent experiments). mCherry is indicated as mCh. Scale bar = 5 microns.

Effects of ΔYSAV on Atg27 localization to the PAS

We next examined whether the tyrosine sorting motif is important for Atg27 transport to the pre-autophagosomal structure (PAS). As previously reported Atg27-GFP colocalized with the PAS marker RFP-Ape1 (Figure 5A) (15, 32). We found 50–55% of the RFP-Ape1 labeled PAS structures had Atg27-GFP during both log phase growth and upon induction of autophagy with rapamycin. Atg27(ΔYSAV)-GFP also localized to the PAS (Figure 5A), but, in contrast to the wild type protein, only about 28% of PAS structures contained Atg27(ΔYSAV)-GFP during log phase growth in rich medium (Figure 5C). However, under autophagy inducing conditions (rapamycin treatment) the ΔYSAV mutant protein was mobilized to the PAS as well as wild-type Atg27 (Figure 5C).

Figure 5. Effect of ΔYSAV on Atg27 localization to the PAS.

Figure 5

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(A) Cells with Localization of Atg27-GFP (SL6502) or Atg27(ΔYSAV)-GFP (SL6504) and the PAS marker RFP-prApe1 in wild type cells. (B) Localization of Atg27-GFP (SL6390) or Atg27(ΔYSAV)-GFP (SL6391) and the PAS marker RFP-prApe1 in an atg1Δ background. Scale bar = 5 microns. (C) Quantification of Atg27-GFP and Atg27(ΔYSAV)-GFP localization to the PAS in the WT and atg1Δ cells during vegetative growth and rapamycin treatment. Each experiment (n≥20 cells) was carried out three times. Not all cells presented have a labeled PAS, as the PAS marker is plasmid-borne. Micrographs are single-plane images from a z-stack. (D) Autophagy was measured in WT (SL 6386), atg1Δ (SL6387), atg27(ΔYSAV) (DYSAV, SL 6388), and atg27Δ (SL 6389) cells using the GFP-Atg8 processing assay. The generation of free GFP (lower band) upon autophagy induction by nitrogen starvation serves as a measure of autophagy function (E) prApe1 processing was measured in WT (SL1463), atg27(ΔYSAV) (SL 5847), apm3D (SL 1652), atg27Δ (SL 6022), and atg1Δ (SL 6320) cells during logarithmic growth.

The fact that autophagy induction by rapamycin did not restore vacuolar membrane localization of the ΔL, ΔYSAV or ASAV mutants (Supplemental Figure S2) suggested that Atg27 vacuolar localization is not dependent on traffic through the PAS. To confirm this, we examined Atg27 localization in an atg1Δ mutant, where many Atg proteins are unable to traffic out of the PAS and accumulate or become trapped there (33). Consistent with previous studies (15), nearly 100% of RFP-Ape1-positive PAS structures accumulated Atg27-GFP in the atg1Δ background, and this was not affected by the ΔYSAV mutation (Figure 5B,C). Noteworthy is that WT Atg27 was still found at the vacuolar membrane and other puncta in the atg1Δ mutant (Figure 6B,C). Therefore, blocking exit from the PAS does not prevent Atg27 delivery to the vacuolar membrane or traffic to other membrane compartments, likely the TGN and endosomes.

Figure 6. Effect of ΔYSAV on Atg9 localization.

Figure 6

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(A) Atg27-mCherry (SL 7075) and Atg27(ΔYSAV)-mCherry (SL 7077) partially localize to Atg9-2x-GFP positive structures. Bar graph indicates the % of Atg9-2x-GFP structures containing Atg27-mCherry (SL 7075) or Atg27(ΔYSAV)-mCherry (SL 7077) (n ≥ 200, combined from 3 independent experiments). mCherry is indicated as mCh. Scale bar = 5 microns. (B) Cells with localization of Atg9-2x-GFP and the PAS marker RFP-prApe1 in wild type (SL 7088), atg27Δ (SL 7089), and atg27ΔYSAV (SL 7090) cells. Bar graph indicates the % of PAS structures containing Atg9-2x-GFP with or without rapamycin treatment to induce autophagy. Each experiment (n≥20 cells) was carried out three times. Not all cells presented have a labeled PAS, as the PAS marker is plasmid-borne. Micrographs are representative single-plane images from a Z-stack. (C) Induction of autophagy in cells expressing Atg9-2x-GFP in the atg27Δ (SL 7060), atg27ΔYSAV (SL 7079) or apm3Δ (SL7091) backgrounds, but not the WT (SL7047) results in GFP fluorescence accumulation in the vacuolar lumen. Bar graph indicates the percent cells showing luminal GFP fluorescence (combined data from at least 2 independent experiments, n≥70 cells).

We also examined whether the YSAV sequence is required for Atg27 to function in autophagy using the GFP-Atg8 processing assay (34). Atg8, a ubiquitin-like protein, is recruited to the growing autophagosome and delivered, together with the autophagosome contents, to the vacuolar lumen (35). In the autophagy reporter assay, once GFP-Atg8 reaches the vacuolar lumen it is cleaved releasing free GFP. GFP-Atg8 processing is evident soon after starvation (1h) in ATG1 WT cells and is blocked in atg1Δ (Figure 5D) (15, 36). Consistent with prior reports, in atg27Δ cells GFP-Atg8 processing was slowed and did not become evident until later times after starvation (2–3h) (Figure 5D & (15)). However, in atg27(ΔYSAV) cells processing of GFP-Atg8 upon autophagy induction was similar to the WT.

We also examined the autophagy-related cytoplasm-to-vacuole transport (Cvt) pathway, since Atg27(ΔYSAV) was observed at the PAS less frequently than the WT protein during log phase growth where Cvt is functional. This pathway can be monitored by the vacuolar delivery and processing of the Cvt cargo precursor aminopeptidase I (prApe1). While atg27Δ exhibited a partial processing defect, consistent with its known phenotype (15), prApe1 was processed into the mature form in atg27(ΔYSAV) cells as efficiently as WT (Figure 5E). In addition, the apm3Δ mutant, which prevented Atg27 localization to the vacuole, had no Cvt phenotype. Collectively, these findings indicate that the Atg27 tyrosine sorting motif and vacuole membrane localization are not essential for Atg27 function in Cvt or autophagy.

Role for the Atg27 tyrosine sorting motif in Atg9 localization

Atg27 is important for trafficking of Atg9, the major autophagy-associated membrane protein (5, 6, 15, 16). Therefore we examined whether deletion of the Atg27 YSAV sorting motif affects trafficking and localization of Atg9. First we compared localization of Atg27-mCherry or Atg27(ΔYSAV)-mCherry with that of Atg9-2xGFP. We found that 33–43% Atg9 puncta exhibited Atg27 fluorescence, with no significant difference between WT Atg27 and Atg27(ΔYSAV) during growth in either rich medium or autophagy-inducing conditions (Figure 6A).

To assess whether deletion of the YSAV motif affects delivery of Atg9 to the PAS, we examined cells expressing Atg9-2xGFP and the PAS marker RFP-Ape1. As shown previously (15), Atg9 delivery to the PAS was severely impaired in the atg27Δ background (≤10% of PAS structures had Atg9, Figure 6B). However, cells expressing Atg27(ΔYSAV) were able to target Atg9 to the PAS as efficiently as WT Atg27 during both logarithmic growth and autophagy induction (Figure 6B). The same results were obtained when Atg8 was used as a PAS marker instead of Ape1, validating that Atg9 was at PAS structures (data not shown).

Yamamoto and colleagues recently showed that atg27Δ cells devoid of PAS structures due to additional gene deletions (atg11Δ atg17Δ) exhibit mislocalization of Atg9 to the vacuolar lumen upon autophagy induction (5). When we examined Atg9 localization in atg27Δ cells with intact PAS structures (ATG11 ATG17) we observed a similar mislocalization of Atg9 under autophagy inducing conditions (Figure 6C). This indicates that the reported mislocalization of Atg9 in atg27Δ atg11Δ atg17Δ (5) was not dependent on a defect in PAS biogenesis. We also observed accumulation of Atg9 in the vacuolar lumen of atg27(ΔYSAV) cells during autophagy (Figure 6C). Thus the YSAV mutation does not prevent Atg9 transport to the PAS (Figure 6B), but selectively affects other post-Golgi transport pathways of Atg9. Similar mislocalization of Atg9 was observed in an apm3Δ mutant (Figure 6C) suggesting that blocking vacuolar localization of Atg27 contributes to this effect.

To further examine the pathway that Atg9-2x-GFP takes to the vacuolar lumen in the atg27Δ and ΔYSAV mutants, we combined these mutations with vps4Δ to block transport through the MVB/PVC. vps4Δ prevented delivery of Atg9-2x-GFP to the vacuolar lumen for either atg27 mutant, resulting in labeling of 85–95% of class E compartments upon rapamycin treatment. In contrast, limited (<20%) of class E endosomes accumulated Atg9 in vps4Δ ATG27 cells, indicating that normally little Atg9 transits the late endosome (Figure 7A, B). Thus mutation of the Atg27 tyrosine sorting motif alone allows Atg9 transit through the endosomal system and MVB directed delivery to the vacuole lumen, even though transport of Atg9 to the PAS is normal in atg27(ΔYSAV) cells. This further supports an endosomal sorting function of Atg27 for Atg9. Interestingly, many vps4Δ cells with atg27 defects also accumulated Atg9-2x-GFP on the vacuolar membrane, although this was more frequent in the ΔYSAV mutant (74%) than in atg27Δ (45%) (Figure 7A, C) (see Discussion).

Figure 7. Deletion of either ATG27 or its YSAV in a vps4Δ background results in autophagy-dependent Atg9 accumulation at the class E compartment and at the vacuolar membrane.

Figure 7

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Strains are: Atg9-2x-GFP vps4Δ (SL7092), Atg9-2x-GFP atg27Δ vps4Δ (SL7093), Atg9-2x-GFP atg27(YSAVΔ) vps4Δ (SL7094). Cells were stained with FM4-64 to mark the vacuolar membrane and the class E compartment. Autophagy was induced by treatment with rapamycin for 6 hours. Scale bar = 2.5 microns. Bar graphs indicate either the percent cells showing Atg9-2x-GFP fluorescence accumulation at the class E compartment (B) or at the vacuolar membrane (C) (combined data from at least 2 independent experiments, n≥50 cells).

DISCUSSION

Our studies have uncovered new information about the trafficking of Atg27 and provided novel insight into its role in Atg9 transport. In addition to localizing to the PAS, Golgi and Atg9-positive structures (15), Atg27 traffics to early and late endosomes and is found on the vacuolar membrane. Consistent with our results, vacuolar membrane localization of Atg27 was reported in a proteomic analysis (37). We found that Atg27 transport from the TGN to the vacuole membrane is direct via the alternate heterotetrameric adaptor AP-3 pathway, and is mediated by a tyrosine sorting motif (YSAV) in the last four amino acids of the Atg27 cytoplasmic tail. The tyrosine sorting signal is also required for the normal distribution of Atg27 in the TGN/endosomal system, but sorting of Atg27 to the PAS is completely normal without the YSAV. We found a large increase in Atg27(ΔYSAV) co-localization with TGN (Sec7) and early endosome (Tlg1) compartments as compared to wild type Atg27, while the percentage of late endosomes marked by Vps27 and Atg27(ΔYSAV) was similar to wild type Atg27. The increased colocalization of Atg27(ΔYSAV)-GFP with Sec7 could reflect its defective AP-3 dependent exit from the TGN, and consequent missorting into early endosomes. However, we did not see an increase in Atg27(ΔYSAV)-GFP in late endosomes or the vacuole. Thus we believe that Atg27 retrieval pathways from late endosomes are operational, while the efficiency of recycling from the early endosome back to the Golgi appears impaired in the tyrosine sorting motif mutant.

Work by others has shown that Atg27 and an associated peripheral membrane protein, Atg23, are important for Atg9 anterograde transport from the TGN to the PAS (5, 15, 16). They play an important role in sorting of Atg9 at the TGN into small mobile transport vesicles destined for the site of autophagosome formation (5). In atg27 or atg23 null mutants devoid of PAS structures, a significant amount of Atg9 was found to traffic through the late endosome/MVB and was delivered to the vacuole lumen during autophagy (5). It was proposed that this mislocalization resulted from missorting of Atg9 due to overexpression or rerouting of Atg9 from the TGN into the endosomal system when Atg23- and/or Atg27-mediated formation of Atg9 transport vesicles was impaired (5). However, we found that Atg9 is also sorted through the MVB and to the vacuole lumen in atg27-(ΔYSAV) cells, a mutant background that had no effect on delivery of Atg9 to the PAS. This is more consistent with an additional role for Atg27 in sorting of Atg9 within the TGN/endosomal compartments. Also, the continued PAS delivery of Atg27 and Atg9 in cells expressing Atg27(ΔYSAV), may partially explain why we saw little effect on autophagy-related processes.

Other work indicates that TGN/endosomal cycling is a normal itinerary for Atg9 in both yeast and animal cells (8, 10–14). In yeast, mutations in several factors mediating shuttling between these compartments can reroute or block Atg9 trafficking in endosomal compartments (10, 11, 38). Likely some of the endosomal Atg9 structures (which may contain Atg27) represent tubulovesicular reservoirs of Atg9 that can be mobilized for autophagy (8, 10). We note that though blocking anterograde and retrograde pathways between endosomes and the TGN affects autophagy and the related-Cvt pathway, in some cases the effects are not discernable or are modest because alternative endosomal pathways can compensate or take over (10, 11). For example both early endosome and late endosome recycling of Atg9 to the TGN must be blocked to observe an effect on starvation-induced autophagy (10). Atg27 may only function in one leg of the TGN/endosomal transport pathways to facilitate Atg9 shuttling, while other compensatory pathways continue to operate. This could also explain why deleting the YSAV sorting motif had no obvious effect on autophagy and Cvt. The Atg27 YSAV might interact with not only AP-3, but with other adaptors that bind tyrosine sorting motifs at the level of the endosome. A candidate is the heterotetrameric adaptor AP-1, which is thought to function in TGN-early endosome traffic (39). A role for AP-1 in autophagy has been reported in animal cells (40).

Overall our results indicate that Atg27 not only plays a role in formation of Atg9 transport vesicles destined for the PAS, but it is important for maintenance of Atg9 TGN/endosomal reservoirs that can be mobilized for autophagy. A final question that then arises is what is the function of Atg27 at the vacuole membrane? A possibility is that Atg27 at the vacuole plays a role in Atg9 maintenance as well, possibly in recycling Atg9 after autophagosome fusion. We observed that atg27Δ vps4Δ and atg27(ΔYSAV) vps4Δ mutants accumulated Atg9, not only in the class E compartment (late endosome), but also at the vacuolar membrane. The accumulation of Atg9 at the vacuolar membrane in these mutants might merely result from the class E defect, as there are other cases where membrane proteins that get stuck in this aberrant LE leak onto the vacuolar membrane (41). However, there are hints for a more direct role at the vacuole. First, apm3Δ, which blocks the AP-3 pathway, also caused accumulation of Atg9 in the vacuole lumen. Yet this adapter mutation should not impair the potential for normal Atg27-mediated recycling of Atg9 from the endosome or Atg9 transport to the PAS. Why Atg9 was delivered to the vacuole lumen in an apm3Δ mutant is still not clear. Furthermore, the vacuolar membrane accumulation of Atg9 was much more extensive in the atg27(ΔYSAV) vps4Δ mutant than in atg27Δ vps4Δ. In the latter, delivery of Atg9 to the autophagy pathway was more severely impaired, so we might have expected more Atg9 to be diverted through the MVB pathway and more vacuole membrane accumulation of Atg9 in vps4Δ atg27Δ than in vps4Δ atg27(ΔYSAV) where PAS-directed Atg9 was normal.

The hypothesis that Atg27 promotes recycling of Atg9 from the vacuole (or autophagosomes) is based upon the observation that Atg9 is present on the outer leaflet of the autophagosome, but is not seen on the vacuolar membrane after autophagy induction and autophagosome fusion (5). Surprisingly, in the atg27 mutants alone, we did not observe accumulation of Atg9 at the vacuolar membrane, but rather Atg9 collects in the lumen of the vacuole. It is a possibility that there is not adequate Atg9 fluxing through the autophagy pathway for visualization on the vacuole membrane even in atg27(ΔYSAV) or apm3Δ, where this proposed recycling pathway would be blocked. Indeed, it has been shown that only a few Atg9 vesicles are needed for a single round of autophagosome formation (5). Another possibility is that if excess Atg9 arrives at the TGN, Atg27 can divert it away from the endosome/MVB pathway to the vacuole membrane via the AP-3 pathway. Then Atg27 could retrieve Atg9 (along with Atg9 from autophagosome fusion) from the vacuole membrane back to the endosome system. Further studies will be required to determine whether Atg27 has any such additional roles in Atg9 trafficking. Also, though Atg27 is a yeast-specific protein, it seems likely that an analogous factor may exist in other eukaryotes to mediate such Atg9 trafficking pathways, and their identification is awaited.

Finally, Atg27’s function has been most studied in the context of autophagy, but it remains possible that it has an autophagy-independent function. Of interest, the large Atg27 luminal region is related to the mannose 6-phosphate receptor homology (MRH) domain family of proteins (42, 43). This suggests that Atg27 may bind N-glycans and could be involved in sorting of glycosylated cargo in the endomembrane system. This sorting may also depend on the Atg27 YSAV motif. Further studies will be needed to explore the role of the Atg27 luminal domain.

MATERIALS AND METHODS

Yeast strain construction and growth

Standard methods and media were employed for genetic manipulations, growth, and transformation of yeast (44). S. cerevisiae strains used in this study are listed in Supplemental Table S1. Unless otherwise indicated, the Longtine method was used for generating the fluorescently-tagged reporters and deletion mutants (18).

To generate the strains marked with Sec7-DsRed, the YIplac204-T/C-SEC7-DsRed.T4 plasmid (gift from Benjamin Glick, University of Chicago) was digested with Bsu36I and transformed into strains SL5837 and SL5845 for integration into the trp1 locus.

Plasmids

The plasmids used in this study are listed in Table S2. To generate the Y268>A (YSAV to ASAV) mutation in Atg27, the QuikChange (Stratagene) method for site-directed mutagenesis was performed on pRS416-ATG27-GFP to generate pRS416-atg27(ASAV)-GFP. pRS416-ATG27-GFP (from J. Nunnari, UC Davis) encodes a 5 residue Gly-Ala linker (AAAAG) between the protein and the fluorescent tag.

Microscopy and image analysis

Vital staining of the vacuolar membrane was performed as described previously by other (45). In short, cells at log phase were incubated in synthetic medium containing 16 μm FM4-64 (Molecular Probes, T3166) or SynaptoRed (CalBiochem, 574799) for 30 minutes at 30°C and then washed twice with fresh medium without dye. A chase period of approximately 2 hours followed, after which cells were imaged as described below.

To induce autophagy for microscopy experiments, log phase cells were incubated in nitrogen starvation medium (SD-N) or synthetic medium containing rapamycin (LC Laboratories, R-5000) at 0.2 μg/mL for the indicated amount time at 30°C (46).

To prevent actin polymerization and ultimately inhibit endocytosis, log phase cells were treated in synthetic medium supplemented with 200 μM LAT-A (Enzo, BML-T119) for 2 hours at 30°C (47). Control cells were treated in medium containing an equal volume of dimethyl sulfoxide (DMSO) as had been used to dilute the LAT-A.

Microscopy was carried out on an Olympus fluorescence BX61 upright microscope equipped with Nomarski differential interference contrast (DIC) optics, a Uplan S Apo 100× objective (NA 1.4), a Roper CoolSnap HQ camera, and Sutter Lambda 10-2 excitation and emission filter wheels, and a 175 watt Xenon remote source with liquid light guide. Image capture was automated using Intelligent Imaging Innovations Slidebook 4.01 for Mac. For all the cells analyzed, a series of optical Z-sections (0.25 μm) were captured. Prior to analysis the stacks were deconvolved using the nearest neighbor algorithm. Representative single-plane micrographs from cells at log phase were chosen to be included in the figures. Note that sometimes Z-planes displaying the best vacuole profiles did not reveal smaller organelles as readily, and vice versa, so in some cases selected planes were chosen based on the organelle marker of interest.

To quantify colocalization of Atg27 with specific organelle markers or reporters, including Atg9-2xGFP, deconvolved Z-stacks were examined to confirm that both fluorescent signals were in the same plane, and that peak fluorescence overlapped in corresponding sections. For PAS (RFP-Ape1) or TGN/endosomal structures (Sec7-DsRed, mCh-Tlg1, GFP-Vps27, and Atg9-2xGFP) results were expressed as the percent of organelle structures that contained the XFP-tagged Atg27 construct. For vacuolar membrane localization, results were expressed as percent of cells in which WT or mutant Atg27-GFP was localized to the rim of the vacuole (stained with FM4-64 or SynaptoRed). Structures, like puncta, on or adjacent to the vacuolar membrane that did not rim the vacuole were not scored as vacuolar membrane localization. To quantify vacuolar lumen localization of Atg9-2xGFP in the WT and mutant backgrounds, cells were stained with FM4-64 or SynaptoRed and results were expressed as percent of cells in which accumulation of green fluorescence in the vacuolar lumen was observed. Similarly, to quantify the amount of class E compartments containing Atg9-2xGFP in cells with the vps4Δ mutation, cells were stained with FM4-64 or SynaptoRed and results were expressed as percent of class E compartments displaying accumulation of Atg9-2xGFP.

Biochemical methods

Ape1 Processing Assays

Strains of interest were grown to log phase in rich media and harvested at the indicated timepoints. Cell lysis was performed as described previously (48). In summary, 1 × 108 cells were resuspended in 400 μL cold extraction buffer (20 mM MES pH 7.0, 1% SDS, 3 M urea, 0.1% SDS, and 50 mM sodium phosphate) supplemented with protease inhibitors. Cells were lysed with 0.45 g of glass beads in 2.0 mL flat-bottom microfuge tubes by vortexing five times for 60 seconds at maximum speed on a vortex genie mixer with icing in between. Samples were then prepared for analysis by SDS-PAGE by addition of loading buffer and then heated for 10 minutes at 77°C. Sample material equivalent to 1 × 107 cells was analyzed by SDS-PAGE and immunoblotting. prApe1 and mApe1 were detected using anti-Ape1 antibodies, kindly provided by Y. Ohsumi (5). Pgk1 was used as a loading control and was detected using anti-Pgk1 monoclonal antibodies (1:1000, Molecular Probes, 459250).

GFP-Atg8 Processing Assays

Strains transformed with pGFP-Atg8 were grown to log phase in C-Ura medium and then shifted to synthetic dextrose minus nitrogen (SD-N) medium to induce autophagy by nitrogen starvation. Aliquots of cells were harvested at the indicated times, lysed, and analyzed by immunoblotting. For each time point, 1 × 108 cells were resuspended in 300 μL RIPA buffer (50mM TrisHCl pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1mM sodium pyrophosphate, and 150mM NaCl) supplemented with protease inhibitors (49). Cells were lysed with 0.45 g of glass beads in 2.0 mL flat-bottom microfuge tubes by vortexing five times for 30 seconds at maximum speed on a vortex genie mixer with icing in between. Samples were spun in a microcentrifuge at top speed for 5 minutes at 4°C and then heated for 5 minutes at 95°C. Sample material equivalent to 2 × 107 cells was analyzed by SDS-PAGE and immunoblotting. GFP was detected using anti-GFP monoclonal antibodies (1:5000, Roche Applied Sciences, Catalog Number 11814460001). Pgk1 was used as a loading control and was detected using the anti-Pgk1 monoclonal antibodies described above.

Immunoblots were analyzed using an Odyssey Infrared Imaging System (LiCor) utilizing appropriate IRDye700 or IRDye800 conjugated secondary antibodies (Rockland).

Supplementary Material

SYNOPSIS.

The transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein, Atg9, to the pre-autophagosomal structure (PAS). This new study demonstrates that Atg27 has a tyrosine-sorting motif in its cytoplasmic tail that mediates its localization to the vacuole membrane by the AP-3 adaptor pathway. Moreover, this motif is also important for preventing Atg9 delivery to the vacuole lumen via the MVB pathway, suggesting that Atg27 retains Atg9 in endosomal reservoirs for mobilization during autophagy.

Acknowledgments

We thank D. Klionsky, G. Odorizzi, D. Katzmann, J. Nunnari, B. Glick, J. Gerst, and Y. Ohsumi for reagents, plasmids and strains. This work was supported by National Institute of Health grant R01-GM055796 to S.K.L. and T32-HL07188 to V.A.S.

ABBREVIATIONS

TGN

trans-Golgi Network

GFP

Green Fluorescent Protein

RFP

Red Fluorescent Protein

PAS

Pre-Autophagosomal Structure

Cvt

Cytoplasm-to-Vacuole Targeting Pathway

ALP

Vacuolar Alkaline Phosphatase

MVB

Multivesicular Body

PVC

Prevacuolar Compartment

LE

Late Endosome

LAT-A

Latrunculin A

WT

wild type

Footnotes

AUTHOR’S COMPETING INTERESTS

The authors declare that they have no competing interests.

CONTRIBUTIONS

VAS and SKL designed the experiments. VAS and DRB performed the experiments. VAS, DRB and SKL analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.

SUPPLEMENTAL MATERIALS

The studies above are supported by Supplemental Figures S1–S3 and Supplemental Tables 1–2.

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Supplementary Materials

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