The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER

SUMMARY

Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration mediated by the Sar1 GTPase and further stabilized by assembly of a multilayered complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the efficiency of this process is dramatically reduced. Specifically, secretory cargoes are retained nearly five times longer at ER subdomains when Sar1 is depleted, but they ultimately remain capable of being translocated to the perinuclear region of cells. Taken together, our findings highlight alternative mechanisms by which COPII promotes transport carrier biogenesis.

In brief

In this study, Kasberg et al. demonstrate that the Sar1 GTPase only regulates the kinetics of anterograde protein trafficking from the ER but is not required for this pathway. These findings highlight the existence of a previously uncharacterized, alternative mode of secretory protein transport between the ER and the Golgi.

Graphical Abstract

INTRODUCTION

In eukaryotic cells, compartmentalization of biochemical processes within membrane-bound organelles improves their efficiency but necessitates the existence of trafficking pathways to move proteins, lipids, and other factors between subcellular locations.^1–5 The oxidative environment of the endoplasmic reticulum (ER) lumen, together with its high concentration of molecular chaperones, facilitates co-translational folding of newly synthesized secretory cargo proteins, which are selectively packaged into coat protein complex II (COPII)-dependent transport carriers that leave the ER from well-organized membrane subdomains.^6–10 Loss of COPII function is incompatible with cell survival and development, consistent with its essential role in regulating the movement of thousands of substrates.^11–22 However, the mechanisms by which COPII coordinates ER membrane remodeling and subsequent cargo trafficking remain poorly defined, particularly in the case of mammalian cells.

Based on a combination of in vivo and in vitro data, there currently exist several overlapping models to describe COPII-mediated protein transport.^10,23–25 Perhaps most strikingly, cryoelectron microscopy (cryo-EM)-based imaging of COPII budding sites has confirmed that biosynthetic cargoes leave the ER via nascent membrane buds, which are linked to an elaborate array of vesicular-tubular membranes that are stably associated with ER subdomains.²⁵ While some literature refers to these tightly juxtaposed organelles collectively as “ER exit sites,” others have distinguished these compartments based on their unique biochemical environments, dubbing the network of ER-adjacent membranes as ER-Golgi intermediate compartments (ERGICs).^26–28 Superresolution fluorescence microscopy has defined at least three unique membrane domains that exist in this early portion of the secretory pathway, delineated by the spatially distinct enrichment patterns of several COPII-associated proteins. Specifically, ER subdomains are marked by the Sar1 guanine nucleotide exchange factor (GEF) Sec12, the large scaffolding protein Sec16a that plays a regulatory role in COPII assembly, and cargo receptors in the Tango1/cTAGE5 family.^8,29–33 Components of the COPII complex accumulate directly adjacent to these regions of the ER, together with TFG, which functions to tether transport intermediates prior to their fusion with one another or neighboring ERGIC membranes.^22,34 ERGICs exhibit elevated concentrations of the lectin ERGIC53, the Rab1 GTPase, and components of the COPI coat, which play an essential role in the anterograde trafficking of secretory cargoes to the Golgi, as well as retrograde transport to the ER.^{25–28,35–39} The entire interface between the ER and the ERGIC is limited to only 300–500 nm in most mammalian cell types, with a high density of membranes present to enable rapid but selective cargo sorting.³⁹

To promote transport carrier biogenesis and cargo export from the ER, the COPII coat complex must associate directly or indirectly with membranes. Upon GTP loading, Sar1 stably penetrates the outer leaflet of the ER phospholipid bilayer, promoting membrane remodeling and tubulation.^40–43 Furthermore, Sec23-Sec24 heterodimers that bind to activated Sar1 and complete the inner layer of the COPII coat also interact with several other ER-associated proteins, including transmembrane secretory cargoes and their receptors, members of the Tango1/cTAGE5 family, and Sec16a.^44–47 The assembled inner coat additionally acts as an adaptor layer for nucleation of an outer cage composed of Sec13-Sec31a heterotetramers.^9,23,42 Together, the multilayered COPII coat has been suggested to stabilize the high degree of membrane curvature necessary to generate canonical transport intermediates.^48,49 However, it has been difficult to dissect how individual COPII components contribute to this process, as assembly of coat subunits is highly interdependent.

Since multiple inner COPII coat proteins exhibit the ability to associate directly or indirectly with ER subdomains in cells, via protein-lipid or protein-protein interactions, we developed strategies to determine whether COPII-mediated transport continues in the absence of one or the other. Notably, recent studies have suggested that Sar1 activity is dispensable for the trafficking of some cargoes, including potassium ion channels, and, in the case of immortalized Caco-2/15 cells, unnecessary for growth and proliferation, although mechanisms that allow ER export to continue in the absence of Sar1 remain unclear.^50–52 Using a combination of genetic approaches, we demonstrate that the loss of Sar1 results in the formation of non-canonical transport intermediates that continue to harbor other COPII subunits and mediate the movement of secretory cargoes from the ER. Under these conditions, COPII exhibits behaviors similar to those of phase-separated liquid droplets or gel-like condensates, ultimately coalescing around Golgi membranes but failing to fully dissociate to allow efficient fusion of transport carriers and the delivery of cargoes. In contrast, depletion of Sec23-Sec24 heterodimers blocks cargo export from the ER. Taken together, our data highlight alternative roles for COPII components during membrane remodeling at the ER, potentially in a manner akin to that of early initiators of endocytosis, which undergo liquid-like assembly at plasma membrane subdomains to catalyze membrane deformation.^53–55

RESULTS

Removal of Sar1 fails to halt secretory protein trafficking

Current models suggest that the Sar1 GTPase plays an obligatory role in membrane remodeling necessary for protein transport from the ER.^6,23,40,41 However, with several studies questioning such an absolute requirement,^50–52 we leveraged CRISPR-Cas9-mediated gene editing in human RPE1 cells to functionally inhibit the two mammalian Sar1 isoforms and delineate their contributions to the early secretory pathway. Guide RNAs directed against Sar1a and Sar1b were used to independently generate several clonal cell lines, which harbored insertions and/or deletions that introduced early stop codons and blocked isoform-specific protein production based on immunoblot analysis (Figures 1A, S1A, and S1B). Loss of Sar1a resulted in several changes to the early secretory pathway compared with control cells, including a reduction in the number of ER subdomains harboring Sec16a and Sec24a, although the concentration of Sec24a at those sites was elevated significantly (Figures S1C–S1F). In addition, the number of ERGIC-associated COPI-positive structures was diminished in cells lacking Sar1a relative to control cells (Figures S1D–S1F). Impacts resulting from the deletion of Sar1b were more modest, consistent with its relatively low level of expression compared with Sar1a (Figures S1A–S1F). In neither case was Golgi volume or morphology significantly affected (Figures S1D and S1G). Taken together, these data suggest that Sar1a serves as the dominant Sar1 isoform in RPE1 cells.

Figure 1. COPII-mediated cargo transport continues in the absence of Sar1.

(A) Cartoon depicting the human SAR1A and SAR1B genomic loci. The positions of the gRNAs used during CRISPR-Cas9 editing (red lines) are highlighted, and the sizes of exons (shown as green boxes) are 1/50 that of introns (shown as black lines).

(B) Representative immunoblots of extracts generated from a CRISPR-modified cell line lacking Sar1a and subjected to siRNA-mediated treatments as shown, using antibodies directed against Sar1 and actin. Extracts were generated at the time point indicated following siRNA treatment.

(C) Quantification of the percentage of Sar1 remaining at different time points following Sar1b siRNA treatment (relative to mock siRNA treatment). Error bars represent mean ± SEM (n = 4 biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test.

(D) Spinning disk confocal microscopy was used to image control RPE1 cells and cells lacking Sar1a either in the presence or in the absence of Sar1b, each expressing ss-DsRed following treatment with SLF (50 μM) to induce cargo disaggregation and release from the ER. Representative time-lapse images are shown (n = 15 cells, each condition; at least three biological replicates). Scale bar, 5 μm.

(E and F) Quantification of cargo (E, ss-DsRed; F, ManII-SBP-GFP) accumulation within the perinuclear region (GM130 positive) in the various mutant backgrounds indicated. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to the 0 min time point). See also Figures S1–S3.

Despite attempts to disrupt Sar1b in cells lacking Sar1a, we were unable to recover viable cells that lacked both isoforms, suggesting a requirement for at least one Sar1 paralog to support growth and proliferation. To circumvent this issue and determine the impact of inhibiting all Sar1 activity, we used a pool of two distinct small interfering RNAs (siRNAs) targeting Sar1b in cells lacking Sar1a, which enabled approximately 98% depletion after 72 h of treatment (Figures 1B, 1C, and S1A). Under these conditions, cell proliferation was inhibited and the frequency of cell death was elevated, confirming an essential role for Sar1 function in RPE1 cells (Figures S1H and S1I). However, a clear population of adherent cells lacking Sar1 remained viable, offering a unique opportunity to study secretory protein transport in the absence of this critical GTPase.

To do so, we leveraged three inducible cargo-release systems developed previously, in which secreted proteins are initially trapped in the ER but undergo synchronous anterograde trafficking following the addition of a specific chemical agent. In one case, a mutant form of FK506-binding protein (FKBP) fused to DsRed and an ER export signal (ss-DsRed) is targeted to the ER lumen, where it assembles into large fluorescent aggregates that can be solubilized by the addition of a synthetic ligand of FKBP (SLF).⁵⁶ In another case, a truncated form of the integral membrane Golgi enzyme mannosidase II fused to the streptavidin binding peptide and GFP (ManII-SBP-GFP) was co-expressed with a fusion between the human invariant chain of the major histocompatibility complex and streptavidin (Ii-streptavidin), which is retained in the ER. Addition of biotin enables ManII-SBP-GFP to be released from Ii-streptavidin, allowing its entry into the secretory pathway.⁵⁷ Finally, a tandem repeat of a self-associating mutant of FKBP (4×FM) fused to HaloTag and the integral membrane protein L1CAM (4×FM-HaloTag-L1CAM) was expressed in cells, trapping it in the ER. Only upon release mediated by an inert rapamycin analog (DDS) is it able to accumulate at the plasma membrane.⁵⁸ Importantly, the presence of HaloTag on the extracellular domain of L1CAM allows it to be labeled specifically after it reaches the plasma membrane using a cell-impermeative HaloTag ligand.⁵⁸

In control cells that had not been subjected to CRISPR-Cas9-mediated editing and cells lacking Sar1a alone, both ss-DsRed and ManII-SBP-GFP accumulated at the Golgi rapidly following release from the ER, based on their increased co-localization with GM130 (Figures 1D, 1E, 1F, and S2A). Surprisingly, simultaneous elimination of both Sar1 isoforms similarly failed to block the export of either cargo from the ER (Figures 1D, 1E, 1F, and S2A). In contrast, even partial reduction of Sec23 levels (~75% depletion relative to control cells) fully stalled ER cargo export, despite clear solubilization of ss-DsRed aggregates following addition of SLF (Figures 1E, 1F, and S2B–S2D). These data strongly suggest that Sar1, but not Sec23, is at least in part dispensable for COPII-mediated cargo export from the ER. In agreement with these findings, 4×FM-HaloTag-L1CAM could be readily detected at the plasma membrane of Sar1-depleted cells following its release from the ER, albeit to a lesser degree compared with control cells, based on quantitative fluorescence measurements (Figures S2E and S2F).

To study the movement of a native COPII cargo (ERGIC-53) in the presence and absence of Sar1, we employed a fluorescent pulse-chase experiment that takes advantage of SNAP-tag, a self-labeling protein tag that binds irreversibly to specific dyelabeled ligands.⁵⁹ We used CRISPR-Cas9-mediated genome editing to generate cells that express SNAP-tag-ERGIC-53 from its endogenous locus. Sequential labeling was performed with JF549-SNAP (to decorate all ERGIC-53), followed by JFX646-SNAP (to decorate only the newly synthesized pool of ERGIC-53 following Sar1 depletion). Based on fluorescence imaging, we found that newly synthesized ERGIC-53 translocated to the perinuclear region of cells, even in the absence of Sar1 (Figure S2G), consistent with our findings examining artificial cargoes.

Together, these studies contradict previous conclusions that were based largely on results obtained following overexpression of dominant-negative isoforms of Sar1 (p.T39N and p.H79G) or inhibition of its GEF Sec12, which suggested that Sar1 activity is required for COPII-dependent cargo export from the ER.^{3,31,41,60–64} Indeed, we confirmed the consequences of these perturbations in blocking secretory protein transport, but additionally found that Sar1a (T39N) expression leads to a diminished number of COPII budding sites, Sar1a (H79G) titrates COPII components away from peripheral budding sites on the ER, and depletion of Sec12 leads to diminished levels of Sec16a and Tango1 at ER subdomains (Figure S3). These findings suggest that overexpression of dominant-negative Sar1 isoforms and Sec12 inhibition have indirect consequences that extend beyond the perturbation of Sar1 function, confounding interpretation of the cargo trafficking defects observed under these conditions. By leveraging an alternative strategy, we found that Sar1 is unexpectedly dispensable for cargo export from the ER.

COPII continues to accumulate at ER subdomains in the absence of Sar1

Based on prior work demonstrating an integral role for Sar1 in nucleating COPII complexes,^41,65 we predicted that its absence would result in COPII subunit dispersal in cells. To directly test this idea, we engineered cells using CRISPR-Cas9-mediated genome editing to express Sec23a fused to HaloTag from its endogenous locus (Figure S4A). Depletion of Sec23b in cells homozygous for HaloTag-Sec23a expression did not impair growth or viability, suggesting that the fusion protein is functional (Figure S4B). Following labeling using a JFX646-conjugated HaloTag ligand, we used live-cell imaging to analyze Sec23a dynamics, demonstrating the existence of two populations, which exhibit either long (>5 μm) or short (<5 μm) track displacements over time (Figure S4C). These data indicate that a small fraction of COPII (2.4% on average) moves several micrometers away from ER subdomains, potentially transiting directly to other organelles, while the vast majority remains at the interface between the ER and the ERGIC membranes (Video S1).

Following the initial phase of Sar1 depletion (48 h after siRNA treatment), the total number of Sec23a-positive structures was reduced by more than 2.5-fold compared with control cells (Figures 2A and 2B). At subsequent time points, however, the population of COPII-labeled structures slowly recovered, suggesting an ongoing ability for COPII complex assembly in the absence of Sar1 (Figure 2C). Consistent with this idea, we routinely found that HaloTag-Sec23a and YFP-Sec31a continued to co-accumulate at de novo sites following Sar1 depletion (Figure 2D). Strikingly, long-term tracking indicated that a substantial proportion of these structures grew in size over time when Sar1 was not present, generating spherical particles larger than 0.8 μm³ in volume, which typically harbored TFG (Figures 2A, 2D, 2E, and 2F), a protein suggested previously to undergo liquid-liquid phase separation (LLPS) at the ER/ERGIC interface.^22,66 After several hours, many of these structures eventually coalesced within the perinuclear region in a manner akin to vesicle-mediated cargo transport to the Golgi apparatus (Figures 2A and 2F; Video S2). Treatment with nocodazole (1 μM), a microtubule depolymerizing agent, inhibited this redistribution, suggesting that COPII accumulation near Golgi membranes is partially dependent on the microtubule cytoskeleton (Figures S4D and S4E).

Figure 2. Loss of Sar1 alters the distribution of COPII coat subunits but fails to block their ability to co-assemble.

(A) Genome-edited cells expressing HaloTag-Sec23a and lacking Sar1a (mock transfected) and those depleted of Sar1b for 48 h were imaged live using spinning disk confocal microscopy following labeling with JFX646-HaloTag ligand. Scale bar, 5 μm; inset bar, 2 μm.

(B and C) Quantification of the number of Sec23a-positive sites in cells lacking Sar1a in the presence and absence of Sar1b is shown at various time points following Sar1b depletion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test, relative to mock treatment (B) or the 48 h time point (C).

(D) Cells lacking Sar1a, depleted of Sar1b, and co-expressing HaloTag-Sec23a and YFP-Sec31a were imaged live using spinning disk confocal microscopy following labeling using JFX646-HaloTag ligand. Representative zoomed images are shown (n = 10 cells; at least three biological replicates each). Scale bar, 2 μm.

(E) The volume distribution of Sec23a-positive structures in the absence of Sar1a is shown following 48 h of mock treatment or Sar1b depletion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to mock treatment).

(F) Cells lacking Sar1a (mock transfected) or depleted of Sar1b for 72 h were immunostained using antibodies directed against Sec24a and TFG (shown only in insets). Representative confocal images (maximum intensity projections) are shown, and an arrow indicates co-localization of Sec24a and TFG. Scale bar, 5 μm; inset bar, 2 μm.

(G–I) HaloTag-Sec23a in cells lacking Sar1a, either in the presence or in the absence of Sar1b, were labeled with JFX646-HaloTag ligand and subjected to photobleaching. Fluorescence recovery is depicted for various-sized HaloTag-Sec23a structures (n = 15 cells each; at least three biological replicates each). See also Figures S4–S6.

Using cells lacking Sar1b (following Sar1a depletion), we independently confirmed that the absence of Sar1 leads to the assembly of enlarged, COPII-positive structures that are not found in control cells (Figures S5A and S5B). In addition, much of the inner coat, as detected using antibodies directed against endogenous Sec24a, accumulated in the perinuclear region following 72 h of Sar1 depletion (Figures S5A and S5B). In contrast, the outer COPII coat (Sec31a) largely failed to localize with Sec24a in this area of the cell (Figure S5A). Based on live-cell imaging of YFP-Sec31a following Sar1 depletion, we found that the outer COPII coat associates with enlarged Sec23a-positive structures as they arrive in the perinuclear region of cells, but subsequently undergoes redistribution into the surrounding cytoplasm, reminiscent of a partial uncoating process (Figure S5C; Video S3). Notably, we found no changes in the distribution of ER membranes in Sar1-depleted cells, highlighting their distinct behavior compared with COPII (Figures S5D and S5E).

To better understand how COPII complexes assemble in the absence of Sar1, we considered the wealth of structural information that exists regarding the interfaces between Sar1 and other COPII subunits.^{9,42,46–49,65,67} In particular, previous work indicates that the binding of Sar1 orders a flexible loop within Sec23 known as the “L loop,” which is important for COPII lattice assembly.⁹ In the absence of this interaction, the L loop is predicted to be disordered,⁹ potentially making Sec23 more likely to undergo phase separation. Moreover, Sec23-Sec24 complexes contain several other disordered domains that likely contribute to their ability to undergo phase transitions in cells.^68–70 Using lattice light sheet imaging, we found that HaloTag-Sec23a endogenously expressed in cells lacking Sar1 exhibited gel-like and liquid-like characteristics, including an ability to undergo cycles of partial dissolution and coalescence during its lifespan (Figure S6A; Videos S4 and S5). This behavior is similar to that seen with the well-characterized P-body component DDX6,⁷¹ as well as the stress-granule marker G3BP1, which undergoes phase separation following treatment with sodium arsenite⁷² (Figures S6B and S6C). Analogous to other liquid-like droplets, COPII condensates were also sensitive to 1,6-hexanediol, which is believed to disrupt weak hydrophobic interactions important for LLPS,⁷³ but phase-separated structures re-formed following washout of the aliphatic alcohol (Figures S6D–S6F).

Leveraging fluorescence recovery after photobleaching (FRAP) studies, we showed that HaloTag-Sec23a molecules exchange rapidly between condensed droplets and the cytoplasm (Figure 2G). The half-time to recovery varied modestly depending on the size/intensity of structures, with those smaller than 0.2 μm³ in volume requiring 3.2 s (±0.3 s) (Figure 2H), while condensates larger than 0.2 μm³ in volume required 4.3 s (±0.4 s) (Figure 2I), precisely the same as that reported previously for overexpressed GFP-Sec23a.⁷⁴ By comparison, HaloTag-Sec23a expressed at endogenous levels in control cells exhibit a half-time to recovery of 2.0 s (±0.5 s). These data suggest that the concentration of Sec23 at unique sites may affect its recovery kinetics. Despite these differences, the mobile fraction of HaloTag-Sec23a was similar under all conditions examined, indicating that the majority of Sec23 is readily exchangeable with the cytoplasmic pool, irrespective of the presence or absence of Sar1 (Figure S6G). This finding is contrasted by the impact of disrupting early secretory pathway function using the fungal metabolite brefeldin A (BFA), which inhibits COPI assembly independent of Sar1.⁷⁵ Specifically, in the presence of BFA (5 μg/mL), a subset of COPII-associated structures becomes modestly enlarged, but their dynamics are significantly altered, exhibiting an increased half-time to recovery (8.2 ± 1.1 s), as well as a reduced mobile fraction (Figures S6G and S6H). These data indicate that COPII condensates formed in the absence of Sar1 are unique compared with structures that assemble as a direct result of COPI inhibition.

COPII condensates associate with cargo released from the ER in the absence of Sar1

To examine whether cargo released from the ER in Sar1-depleted cells associates with COPII, we employed superresolution stimulated emission depletion (STED) microscopy. Specifically, following 60 h of siRNA treatment targeting Sar1b, cells lacking Sar1a and expressing ss-DsRed were exposed to SLF and processed for immunofluorescence-based imaging using antibodies directed against Sec24a. Under these conditions, we found that released cargo associated directly with COPII condensates prior to reaching the perinuclear region of cells, strongly suggesting that at least a subset of these structures represents bona fide transport intermediates (Figure 3A). Consistent with this idea, labeling of Sar1-depleted cells expressing native levels of HaloTag-Sec23a with a lipophilic carbocyanine dye that indiscriminately labels intracellular lipid bilayers confirmed the presence of membranes with COPII condensates (Figure S7A). We also conducted a series of live-cell imaging experiments to further demonstrate that secretory cargoes leave the ER in association with COPII condensates in cells lacking Sar1. To do so, we used CRISPR-Cas9-mediated editing to append GFP onto the amino terminus of the Sec61 translocon β subunit (Sec61 β), enabling visualization of the ER network while monitoring HaloTag-Sec23a and a releasable cargo (ss-DsRed) (Figure S7B). ER dynamics were analyzed by tracking spaces between Sec61 β-labeled tubules and sheets⁷⁶ in the absence of Sar1, following the addition of SLF. Using single-particle tracking under these conditions, we found that the displacement of COPII condensates and ss-DsRed dramatically exceeded that of ER membranes (Figure 3B). These data suggest that the translocation of cargo-associated COPII condensates cannot be explained simply by redistribution of the ER. Moreover, direct examination of individual cargo trafficking events further supported this conclusion, demonstrating that ss-DsRed moves away from ER membranes toward the perinuclear region of cells in association with COPII condensations when Sar1 is absent (Figures 3C, S7C, and S7D). Fluorescence intensity measurements indicated that ss-DsRed concentrated at ER subdomains where COPII condensates were present, but only until a displacement event (greater than 1 μm) occurred, after which levels of ss-DsRed remained constant as carriers moved toward the cell center (Figure 3D).

Figure 3. COPII condensates associate with secretory cargoes that leave the ER in the absence of Sar1.

(A) Cells expressing ss-DsRed and lacking both Sar1 isoforms were immunostained using antibodies directed against Sec24a and GM130 following treatment with SLF (50 μM) and imaged using STED microscopy. Representative images are shown (n = 15 cells; at least three biological replicates). Scale bar, 2 μm.

(B) Cells expressing EGFP-Sec61β and HaloTag-Sec23a in the absence of Sar1 were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following cargo release and labeling with JFX646-HaloTag ligand. Violin plots show the relative displacement of each marker over time (n = 10 cells; three biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test, compared with Sec61β displacement.

(C) Cells lacking both Sar1 isoforms and expressing native EGFP-Sec61β and HaloTag-Sec23a were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following treatment with SLF (50 μM) and labeling with JFX646-HaloTag ligand. Representative images are shown (n = 7 cells; three biological replicates). Arrowheads indicate accumulation of ss-DsRed with COPII condensates, which ultimately move away from their site of origin. Scale bar, 2 μm.

(D) Relative fluorescence intensities of cargo (ss-DsRed) and COPII (HaloTag-Sec23a) were measured at ER subdomains over time. Error bars represent mean ± SEM. An asterisk highlights the time point at which cargo and COPII undergo a >1 μm displacement, and all measurements are aligned with respect to this time point (n = 10 cells; three biological replicates).

(E) Expression of 4×FM-HaloTag-L1CAM was transiently induced in control cells co-expressing Sar1 (H79G) or in cells lacking Sar1a and depleted of Sar1b, each labeled with JFX646-HaloTag ligand, and subjected to photobleaching after treatment with DDS for 60 min. Normalized fluorescence recovery in each case is shown (n = 10 cells each; at least three biological replicates each), and error bars represent mean ± SEM. An asterisk highlights the time point at which individual sites exhibiting elevated 4×FM-HaloTag-L1CAM fluorescence were bleached.

(F) Cells lacking Sar1a and natively co-expressing EGFP-Sec61β and HaloTag-Sec23a in the presence and absence of Sar1b were transfected with a construct encoding ss-DsRed and imaged as described for (B). The length of time ss-DsRed remained associated with HaloTag-Sec23A prior to undergoing displacement (more than 1 μm) was determined in each case. **p < 0.01, calculated using a t test, compared with cells lacking only Sar1a. See also Figure S7.

Collectively, these data suggest that COPII condensates that form when cells are depleted of Sar1 promote cargo loading into nascent transport intermediates, which ultimately undergo scission from the ER. To further validate this idea, we conducted a series of FRAP studies to determine whether cargo released in the absence of Sar1 remained capable of exchanging with the lumenal ER pool remaining there. Specifically, 4×FM-HaloTagL1CAM was released using DDS in cells lacking Sar1 and was subsequently subjected to photobleaching to measure recovery time. Under these conditions, released 4×FM-HaloTag-L1CAM showed an absence of fluorescence recovery, indicating it was no longer exchangeable, consistent with scission from the ER (Figure 3E). By contrast, in cells expressing a dominant-negative isoform of Sar1 (H79G), which inhibits the scission of COPII carriers from the ER,^3,77 HaloTag-L1CAM fluorescence that was presumably concentrated within nascent buds recovered quickly after photobleaching (Figure 3E). Notably, in the absence of Sar1, cargo remained associated with budding sites for an extended period of time compared with control cells, suggestive of a delay in export from the ER (Figure 3F). These studies support a model in which Sar1 is dispensable for cargo export from the ER, but plays a critical role in regulating the kinetics of COPII-mediated anterograde protein sorting and transport.

Previous work that reached a similar conclusion regarding the expendable nature of Sar1 in secretory protein trafficking suggested that the early secretory pathway becomes restructured when Sar1 is limiting, with COPI potentially directing an alternative export route from the ER.⁵² To address this possibility, we conducted a series of quantitative immunofluorescence experiments, revealing that the integrity of ER budding sites marked by Sec16a and Tango1 was not disrupted by the loss of Sar1 (Figures 4A and 4B). However, the fluorescence intensities of both factors were increased significantly under these conditions relative to control cells (Figures 4A–4C), an effect that was mirrored by their higher levels of overall expression (Figures S8A and S8B). Similarly, quantitative imaging of ERGIC-53 and TFG indicated elevated levels of these proteins when Sar1 was absent (Figures 4D and 4E), consistent with immunoblotting studies (Figures S8C–S8E), whereas expression levels of COPII subunits were unaffected (Figures S8E and S8F). Surprisingly, we were unable to detect ERGIC-53 adjacent to ER subdomains harboring Sec16a under these conditions. Instead, in cells depleted of Sar1, ERGIC-53 was concentrated within the perinuclear region of cells (Figure 4D). These data strongly suggest that conventional ERGIC membranes become destabilized and lose their integrity in the absence of Sar1. In agreement with this idea, in cells lacking Sar1, COPI exhibited a largely cytoplasmic distribution (Figures 4F–4H), suggesting a dependence on Sar1-mediated ER membrane remodeling to subsequently load onto ERGIC membranes and drive anterograde transport of cargoes to the Golgi. Together, these data indicate that COPI is unlikely to participate in cargo transport from the ER when Sar1 is depleted in a penetrant manner.

Figure 4. The absence of Sar1 destabilizes ERGIC membranes.

(A, B, D, E, and F) Cells lacking Sar1a (mock transfected or depleted of Sar1b for 72 h) were immunostained using antibodies directed against Sec16a (A), Tango1

(B), ERGIC-53 (and Sec16a shown in insets, with arrows highlighting their juxtaposed distribution) (D), TFG (E), or COPB1 (and GM130 shown in insets) (F). Representative confocal images (maximum intensity projections) are shown. Scale bars, 5 μm; inset bars, 2 μm.

(C) Quantification of the fold change in the fluorescence intensities of Sec31a, Sec16a, and Tango1 in the absence of Sar1 (relative to mock siRNA treatment). Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test.

(G) Representative immunoblots of extracts generated from control and CRISPR-modified cell lines lacking Sar1a and either in the presence or in the absence ofSar1b, using antibodies directed against COPB1 and actin.

(H) Quantification of the fold change in COPB1 levels in the absence of Sar1 (relative to mock siRNA treatment of cells lacking only Sar1a). Error bar represents mean ± SEM (n = 4 biological replicates). See also Figure S8.

Cargo trafficking through the Golgi is delayed in the absence of Sar1

To determine the ultimate fate of cargoes released from the ER following Sar1 removal, we again leveraged live-cell imaging. These studies demonstrated that in the absence of Sar1, COPII-associated cargoes that arrive in the perinuclear region of cells fail to move further through the secretory pathway in an efficient manner, in contrast to control cells (Videos S6 and S7), suggesting that COPII coat components must disassemble from the surface of transport intermediates to enable membrane fusion. To test this concept, we dispersed COPII condensates using 1,6-hexanediol and found that this treatment enabled stalled cargo to transit through the perinuclear region and traffic toward the plasma membrane, strongly suggesting that the transport intermediates, which accumulate in Sar1-depleted cells, are functional with respect to secretory cargo trafficking (Figures 5A, 5B, and S9A; Video S8). In contrast, addition of 2,5-hexanediol, which fails to disperse COPII condensates (Figure S9B), similarly fails to increase the rate of cargo entry into and through the Golgi (Figures 5A and 5B; Video S9).

Figure 5. The absence of Sar1 leads to the formation of COPII condensates that interfere with secretory cargo movement through the Golgi.

(A) Time-lapse spinning disk confocal microscopy was used to image cargo (ss-DsRed) accumulated in the perinuclear region of cells lacking Sar1 following addition of 1,6-hexanediol (top) or 2,5-hexanediol (bottom). Representative images are shown (n = 10 cells; three biological replicates). Scale bar, 5 μm.

(B) Quantification of cargo (ss-DsRed) remaining within the perinuclear region of cells lacking Sar1 following 30 min of incubation with 1,6-hexanediol or 2,5-hexanediol (relative to the 0 min time point). Error bars represent mean ± SEM (n = 10 cells each; three biological replicates). ***p < 0.001, calculated using a t test, compared with treatment with 2,5-hexanediol.

(C) Representative electron micrographs taken within the perinuclear region of high-pressure-frozen cells lacking Sar1a following a mock siRNA treatment or Sar1b depletion. The nuclear envelope (NE) is indicated in each image (n = 5 cells each; at least three biological replicates). Scale bar, 500 nm. See also Figure S9.

To gain a higher resolution view of the impacts resulting from Sar1 depletion, we turned to electron microscopy. In contrast to control cells, which exhibit canonically stacked Golgi cisternae within the perinuclear region, we instead found a dramatic accumulation of spherical membrane-bound compartments that were highly heterogeneous in size in all cells lacking both Sar1 isoforms (Figures 5C and S9C). In addition, the lumen of the ER became distended in cells depleted of Sar1, although the morphology of the nuclear envelope remained largely unaffected (Figure S9C). The distribution of the unusual spherical compartments was similar to that of COPII condensates visualized by fluorescence microscopy, which accumulate in the perinuclear region following Sar1 inhibition. We therefore sought to determine whether the COPII condensates correspond to any known membrane-bound or membraneless organelle. Our findings demonstrated that COPII condensates failed to co-localize with several other proteins known to undergo phase separation, nor did they associate with autophagosomes, lysosomes, or the highly disordered protein Sec16a, which has been implicated in the formation of phase-separated “Sec bodies” following nutrient starvation^69,70 (Figures S9D–S9G). These data suggest that COPII condensates, which form in the absence of Sar1 and accumulate in the perinuclear region of cells, represent unique liquid- or gel-like structures that associate with membrane-bound compartments.

DISCUSSION

Despite intense study over several decades, the mechanisms by which COPII facilitates the anterograde transport of membrane proteins from the ER remain controversial. Classical paradigms supported by elegant biochemical and reconstitution-based studies suggest that COPII remodels lipid bilayers to form cargo-laden, coated vesicles, while more recent studies raise the possibility that COPII may also promote the formation of highly curved membrane tunnels that concentrate secretory cargoes therein, but fail to coat transport intermediates.^23–25 In both cases, COPII is responsible for membrane deformation, a process that has been largely ascribed to the inner coat subunit Sar1, which stably penetrates ER lipid bilayers when bound to GTP, driving membrane tubulation.^40–42,65 In addition, activated Sar1 has been proposed to be the major recruitment factor for Sec23-Sec24 heterodimers, completing the inner layer of the COPII coat complex.^9,42,65 However, our findings indicate that Sar1 is surprisingly not required for secretory protein export from the ER, directly challenging the idea that it is indispensable for membrane bending and cargo loading. In contrast, the inner coat component Sec23 is irreplaceable in these processes, supporting a model in which COPII can function independent of Sar1 to remodel ER membranes, in agreement with previous in vitro studies.⁴¹

This raises the important question of how Sec23-Sec24 continues to be recruited to ER subdomains following Sar1 removal. One possibility is that the scaffolding protein Sec16a, which binds directly to Sec23 and Sec24 and localizes independent of COPII to ER budding sites, partially substitutes for Sar1 in this capacity.^8,44,45 Notably, the levels of Sec16a at ER subdomains are dramatically upregulated in the absence of Sar1, which likely enables enhanced recruitment of the remaining COPII components. In an analogous manner, elevated levels of Tango1, which has also been shown to associate directly with Sec23 at ER subdomains, may further promote the accumulation of inner COPII coat components irrespective of Sar1 presence, and simultaneous binding of Sec24 to secretory cargoes would additionally aid Sec23-Sec24 membrane association.^32,33,46,47 Via these multivalent, but relatively low-affinity interactions, a critical concentration of Sec23-Sec24 heterodimers may be reached to drive their local demixing, creating phase-separated condensates, which are capable of membrane deformation.^53–55 In contrast, depletion of the Sar1 GEF Sec12 fails to promote phase separation of COPII subunits, potentially due to the reduced levels of Sec16a and Tango1 found at ER budding sites under these conditions, limiting the ability of COPII subunits to concentrate locally. Further studies will be necessary to define mechanistically how COPII condensates promote transport carrier biogenesis, but the propensity of COPII components to undergo a phase transition is not unprecedented. During amino acid starvation in Drosophila S2 cells, most COPII subunits, with the notable exception of Sar1, co-assemble to form liquid-like stress assemblies that have been dubbed “Sec bodies,” which are believed to function as storage depots to protect early secretory pathway components from degradation until nutrient conditions improve.⁶⁹ Similar results were recently obtained in mammalian INS-1 cells, which additionally highlighted a key role for Sec16a in promoting Sec body formation.⁷⁰ Although COPII condensates observed following Sar1 inhibition appear distinct from Sec bodies, in terms of both overall composition and function, the ability of Sec23-Sec24 to undergo phase separation appears to be evolutionarily conserved.

Recent studies using cryoelectron tomography and subtomogram averaging have revealed the overall architecture of the COPII complex assembled on a membrane surface, highlighting a multitude of highly structured interactions between layers of the coat, as well as the association of Sar1 with the lipid bilayer.^9,42 Based on this network of associations, models have emerged to suggest how COPII promotes membrane reorganization to support transport carrier formation and cargo trafficking.^9,42 However, the many low-complexity regions found in COPII subunits, particularly those within Sec24 and Sec31 isoforms, remain poorly defined, limiting our understanding of their contributions to this process. Our work demonstrating that COPII continues to facilitate secretory cargo export from the ER in the absence of Sar1 suggests additional roles for the coat in membrane remodeling. In particular, phase separation of COPII while associated with ER membranes may lead to sufficient molecular crowding and steric pressure to promote membrane deformation.^78–80 This idea is in agreement with in vitro studies showing that the large hydrodynamic radii of disordered domains from epsin1 or AP180 are adequate to drive membrane tubulation when either is tethered to a bilayer.^53,54 In addition, the liquid-like state of COPII condensates may also facilitate further membrane remodeling events that enable the release of coated transport carriers from the ER, in a manner similar to that shown recently for Eps15-Fcho liquid droplets during endocytosis.^55,80 Considering our finding that the dynamics of COPII subunits within condensates formed following Sar1 depletion is highly similar to that observed in control cells, our data raise the intriguing possibility that COPII phase separation normally contributes to transport carrier biogenesis and secretory efflux from the ER (i.e., when Sar1 is present). In agreement with this idea, recent work suggested that Sec16a co-phase separates with multiple components of the COPII machinery to facilitate anterograde cargo trafficking.⁸¹ Further studies will be necessary to fully explore this idea and determine how the action of the Sar1 GTPase regulates the equilibrium between assembly of structured COPII lattices and the formation of unstructured COPII condensates.

Despite ongoing protein export from the ER in the absence of Sar1, cells failed to proliferate under these conditions, suggesting other consequences to endomembrane trafficking. Of particular note, Sar1 inhibition resulted in a lack of discernible ERGIC membranes juxtaposed to ER subdomains, a finding consistent with an absence of canonical COPII transport intermediates, which normally uncoat and fuse to generate these compartments.^26–28 Instead, COPII condensates of various sizes that form in the absence of Sar1 appear to mediate secretory protein trafficking directly to the perinuclear region of cells in a manner dependent on microtubules. This idea is supported by previous work showing that Sec23 interacts with the dynactin complex, enabling movement on microtubules toward centrosomes located near the cell center, together with the Golgi apparatus.⁷⁷ However, we found that fusion of non-canonical COPII transport carriers with the Golgi was delayed, impeding cargo entry and resulting in a dramatic accumulation of COPII condensates in the perinuclear region over time. These data suggest an important role for Sar1 GTPase activity in fully disassembling the inner COPII coat ahead of membrane fusion, a function that has been speculated for decades, despite a lack of clear evidence in animal cells.⁸² Alternatively, the absence of Sar1 may lead to the formation of aberrant COPII assemblies, which cannot disassemble efficiently, while the presence of Sar1 leads to the generation of reversible and dynamic COPII complexes that facilitate rapid and efficient cargo sorting and transport. When taken together, our studies highlight several functions of COPII, which likely act throughout the life cycle of ER-derived secretory transport carriers at the ER/ERGIC interface.

Limitations of the study

Although our data strongly support a model in which COPII coat proteins remain capable of directing secretory protein export from the ER in the absence of the Sar1 GTPase, the mechanisms that underlie membrane remodeling events necessary for transport carrier biogenesis under these conditions remain unclear. Future studies leveraging high-resolution correlative light and electron microscopy will likely be necessary to define the precise distribution of COPII condensates during this process. Moreover, reconstituting the activity of COPII condensates on the surface of ER-derived microsomes may help to resolve their role in promoting membrane budding when Sar1 is not present. Due to technical limitations, such studies have not been feasible to perform, limiting our ability to understand whether COPII coat proteins directly or indirectly bind to lipid bilayers and stimulate membrane bending. In addition, a clear understanding of the potential roles that Sec16a, Tango1, and nascent secretory cargoes play in facilitating COPII phase separation at ER subdomains remains to be defined. This is complicated by the significant consequences of Sar1 depletion on overall cellular architecture, leaving open questions around the mechanism by which COPII coat proteins are recruited to ER membranes without Sar1.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anjon Audhya (audhya@wisc.edu).

Materials availability

All reagents generated in this study are available from the lead contact with a Material Transfer Agreement.

Data and code availability

• All original microscopy data reported in this paper will be shared by the lead contact upon request.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

This study uses human hTERT-immortalized RPE1 cells (CRL-4000 from ATCC; sex: female), which have been authenticated using STR profiling analysis.

METHOD DETAILS

CRISPR/Cas9-mediated genome editing, siRNA-mediated depletion, and cell proliferation studies

To generate human RPE1 cells that natively express either HaloTag, EGFP, or SNAP-tag fusion proteins, CRISPR/Cas9-mediated genome editing was used. In the case of HaloTag-Sec23a, a plasmid co-expressing Cas9-GFP and a guide RNA targeting the amino-terminus of Sec23a was electroporated into cells together with a plasmid-based homology directed repair (HDR) template encoding the HaloTag, a linker, and ~800 base pairs of flanking genomic DNA, both upstream and downstream of the Cas9 cut site. Fluorescence activated cell sorting (FACS) was used to identify individual cells that were GFP positive, and homozygous genome edited clones were subsequently validated by fluorescence imaging, immunoblot analysis and Sanger sequencing. To generate cells expressing EGFP-MAP1LC3B and EGFP-Sec61b, synthetic gRNAs were individually co-transfected with HDR templates generated previously by the Allen Cell Institute and recombinant Cas9. Following FACS, positive clones were identified based on GFP fluorescence and validated. In the case of SNAP-tag-ERGIC-53, a synthetic gRNA was co-transfected with an HDR template and recombinant Cas9 into cells using TransIT-X2. Cells were subsequently labeled with JFX646-SNAP ligand and subjected to FACS to identify individual genome edited clones, which were validated as described above.

To introduce lesions into the Sar1a, Sar1b, and Sec23b coding sequences and disrupt their expression, we used gRNAs targeting each, which were expressed from plasmids encoding Cas9-GFP and transfected separately into cells using FuGENE HD. Clones isolated by FACS were examined by immunoblot analysis and subjected to Sanger sequencing to confirm bi-allelic editing.

Depletion studies were conducted using multiple siRNAs, which were transfected individually or in tandem into cells using Lipofectamine RNAiMAX. To study cell viability and death, we used a Viability/Cytotoxicity Assay Kit, which contains two markers, Calcein AM and Ethidium homodimer-III (EthD-III), to label live or dead cells, respectively. We measured and compared the average intensity sum of Calcein-AM and Ethidium homodimer-III (EthD-III) in quadruplicates.

Fluorescence imaging studies and image analysis

Live cell imaging was performed using a Nikon Ti2 spinning disk confocal microscope equipped with a 60x oil immersion objective (1.4 NA) and a Hamamatsu ORCA-Flash4.0 sCMOS camera or a Molecular Devices ImageXpress Micro 4 (IXM4) high content imaging system with a 40x dry objective (0.95 NA) and a sCMOS camera. For ManII-SBP-GFP trafficking, streptavidin (100 μM) was added to the growth media to prevent premature trafficking of cargo. The addition of biotin (200 nM) triggered cargo efflux from the ER. Release of ss-DsRed was mediated by the addition of 50 μM SLF, as described previously,⁵⁶ and its translocation to the perinuclear region of cells was quantified using the spot tracking feature of IMARIS software. Expression of 4xFM-HaloTag-L1CAM was transiently induced in cells overnight using doxycycline (10 nM), and its release from the ER was triggered using DDS (1 μM) in the presence or absence of nocodazole (1 μM). Cell surface labeling of 4xFM-HaloTag-L1CAM was conducted using JF549i-HaloTag ligand (1 nM). For high-speed imaging of HaloTag-Sec23a, GFP-G3BP1, and RFP-DDX6, a 3i Lattice Lightsheet (LLS) microscope was used, outfitted with a Hamamatsu ORCA-Flash4.0 V3 sCMOS camera, a 25x (1.1 NA) water immersion detection objective, and a 28x (0.71 NA) water immersion illumination objective. Spinning disk confocal microscopy and LLS imaging of fluorescent fusions to G3BP1 and DDX6 were conducted following viral-mediated transduction, and all HaloTag-fusions were labeled with dye-conjugated HaloTag ligands (100 nM) overnight prior to washout and imaging. For fluorescent pulse chase studies, cells lacking Sar1a but natively expressing SNAP-tag-ERGIC-53 were labeled sequentially with JF549-SNAP and SF650-SNAP (50 nM each), in the presence and absence of siRNAs targeting Sar1b. Specifically, cells were mock-treated or treated with siRNAs directed against Sar1b for 24 hours prior to labeling with JF549-SNAP (to decorate the entire population of ERGIC-53 in cells). Following dye-labeling (24 hours), cells were rinsed three times with growth media and allowed to efflux unbound dye (3 times; 15 minutes each at 37°C). Cells were then labeled 24 hours later using JFX646-SNAP (12 hours; to decorate newly synthesized ERGIC-53) and imaged after unbound dye was allowed to efflux.

For diffraction-limited immunofluorescence studies, cells were fixed using 4% paraformaldehyde at 37°C for 15 minutes or 100% methanol at −20°C for 20 minutes, followed by permeabilization and antibody labeling (1 μg/ml) at 4°C overnight.⁸² Immunofluorescence studies were conducted using validated antibodies (see key resources table). After thorough washing, coverslips were incubated with secondaries antibodies (AlexaFluor conjugates) for 1 hour and mounted using Vectashield. Imaging datasets were comprised of 25–35 sections separated by 0.3 μm. For stimulated emission depletion microscopy (STED), AbberiorSTAR, Alexa Fluor 555, or Alexa Fluor 594 secondary conjugates were used, prior to mounting using Prolong Diamond Antifade. Coverslips were cured at room temperature in the dark for 24 hours prior to imaging on a Leica TCS SP8 3x super-resolution imaging system using the 775 nm depletion laser. Based on the imaging of diffraction-limited fluorescent beads, the full width at half maximum of the STED point-spread function was determined to be 90 nm. Live super-resolution imaging of COPII condensates was conducted on a Nikon AXR imaging system using a 100x oil immersion objective or a Zeiss LSM 880 with a 63x (1.4 NA) oil immersion objective using the Airyscan super-resolution detector. Data were further processed by Nikon proprietary ‘denoise’ software or deconvolved using automatic Airyscan settings to elucidate their geometry.

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-COPB	Santa Cruz Biotechnology	sc-393615
Anti-Rab1a	Cell Signaling Technology	D3X9S
Anti-Sec16a	Bethyl Laboratories	A300-648A; RRID:AB_519338
Anti-Tango1	MilliporeSigma	HPA055922; RRID:AB_2682971
Anti-GM130	BD Sciences	BD610823
Anti-ERGIC-53	Santa Cruz Biotechnology	sc-66880; RRID:AB_2136001
Anti-Sec24a	Santa Cruz Biotechnology	sc-169279; RRID:AB_10841885
Anti-Sec22b	Schindler et al.64	https://doi.org/10.1073/pnas.0910342106
Anti-Sec31a	BD Sciences	612351; RRID:AB_399717
Anti-RINT1	MilliporeSigma	HPA031646; RRID:AB_2673975
Anti-calreticulin	Enzoscience	ADI-SPA-601; RRID:AB_10630195
Anti-Golgin-97	MilliporeSigma	HPA050555; RRID:AB_2681174
Anti-TFG	Novus Biologicals	NBP2-62212
Anti-Sec23a	ThermoFisher Scientific	PA5-28984; RRID:AB_2546460
Anti-Sec23	ThermoFisher Scientific	PA1-069A; RRID:AB_2301581
Anti-Sec13	Proteintech	15397-1-AP; RRID:AB_2186234
Anti-β-actin	MilliporeSigma	A1978:AB_476692
Anti-Sar1	Schindler et al.64	https://doi.org/10.1073/pnas.0910342106
Chemicals, peptides, and recombinant proteins
TRIzol reagent	ThermoFisher Scientific	15596026
Alt-R® CRISPR-Cas9 tracrRNA	IDT	1072534
FuGENE-HD transfection reagent	Promega	E2311
TransIT-X2 dynamic delivery system	Mirus	MIR 6003
Lipofectamine RNAiMAX transfection reagent	ThermoFisher Scientific	13778150
Streptavidin	Promega	Z7041
Biotin	MilliporeSigma	B4501
SLF	Cayman Chemical	10007974
D/D solubilizer (DDS)	Clontech	635034
Nocodazole	Cell Signaling Technology	2190S
BFA	MilliporeSigma	B7651
Vectashield Mounting Media	Vector Laboratories	H-1000
Prolong Diamond Antifade Mounting Media	ThermoFisher Scientific	P36965
Sodium arsenite	ThermoFisher Scientific	LC229002
1,6-hexanediol	MilliporeSigma	240117
2,5-hexanediol	MilliporeSigma	H11904
JFX650-SNAP ligand	Janelia Research Campus	N/A
JFX650-HaloTag ligand	Janelia Research Campus	N/A
JF549i-HaloTag ligand	Janelia Research Campus	N/A
JF549-SNAP ligand	Janelia Research Campus	N/A
Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 555	ThermoFisher Scientific	A21127
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	ThermoFisher Scientific	A21242
LysoTracker Red DND-99	ThermoFisher Scientific	L7528
DilC18	Biotium	60010
Epon EMbed 812	Electron Microscopy Sciences	14120
Critical commercial assays
SuperSignal West Femto Maximum Sensitivity Substrate	ThermoFisher Scientific	34096
Cell viability/Cytotoxicity Assay Kit	Biotium	30002
High-Capacity cDNA Reverse Transcription Kit	ThermoFisher Scientific	4368814
Experimental models: Cell lines
hTERT-immortalized RPE1 cell line	ATCC	CRL-4000
Oligonucleotides
EGFP-MAP1LC3B gRNA: 5’-TGGTG CAGGGATCTGGGCGG-3’	Allen Institute for Cell Science	N/A
EGFP-Sec61b gRNA: 5’-GCCATAC CATATTGGAGATG-3’	Allen Institute for Cell Science	N/A
SNAP-tag-ERGIC-53 gRNA: 5’-GG TCGCTTCGTCCGGGGCGA-3’	This study	N/A
Sar1a siRNA #1: 5’-GAACAGAUG CAAUCAGUGA-3’	Cutrona et al., 2013	https://doi.org/10.1111/tra.12060
Sar1a siRNA #2: 5’-CCAGUAUAU UGACUGAUGU-3’	Cutrona et al., 2013	https://doi.org/10.1111/tra.12060
Sar1b siRNA #1: 5’-GCAUAACUU GAAUUCAAUA-3’	Cutrona et al., 2013	https://doi.org/10.1111/tra.12060
Sar1b siRNA #2: 5’-CUACCUUCC UGCUAUCAAU-3’	Cutrona et al., 2013	https://doi.org/10.1111/tra.12060
Sec23a siRNA #1: 5’-CAGACUCA UAAUAAUAUGUAU-3’	Hanna et al., 2017	https://doi.org/10.1073/pnas.1709120114
Sec23a siRNA #2: 5’-AUGACGGU UGUAACUACUAAA-3’	Hanna et al., 2017	https://doi.org/10.1073/pnas.1709120114
Sec23b siRNA #1: 5’-CACGUUAC AUCAACACGGA-3’	Hanna et al., 2017	https://doi.org/10.1073/pnas.1709120114
Sec23b siRNA #2: 5’-CACUAUGA GAUGCUUGCUA-3’	Hanna et al., 2017	https://doi.org/10.1073/pnas.1709120114
Sec12 siRNA #1: 5’-CCAGAGCUC CUUGGGUCCCAUGAAA-3’	Saito et al., 2014	https://doi.org/10.1083/jcb.201312062
Sec12 siRNA #2: 5’-CAGACUUUA GCUCCGAUCCACUGCA-3’	Saito et al., 2014	https://doi.org/10.1083/jcb.201312062
GAPDH F (qPCR primer): AGCCAC ATCGCTCAGACAC	Kubota et al., 2015	https://doi.org/10.3892/or.2014.3605
GAPDH R (qPCR primer): GCCCAA TACGACCAAATCC	Kubota et al., 2015	https://doi.org/10.3892/or.2014.3605
Sec16A F (qPCR primer): ATCGTAA GGCAGGAAGTG	This study	N/A
Sec16A R (qPCR primer): CTGATGA CTGGAATGGTTTG	This study	N/A
Recombinant DNA
pUC57 Sec23a CRISPR HDR (5’-CTCCACAATGACAACCTATT-3’)	This study	N/A
pUC57 SNAP-tag-ERGIC-53	This study	N/A
li-Str_ManII-SBP-EGFP	Addgene	65255
piGH-DsRed-Express2-FKBP(LV)(C22V)	Casler et al., 2019	https://doi.org/10.1083/jcb.201807195
Cag 4xFM-HaloTag-L1CAM	This study	N/A
phage UbiC G3BP1-GFP-GFP	Addgene	119950
phage UbiC tagRFP-T-DDX6	Addgene	119947
pTALEN TET Sar1a (H79G)	This study	N/A
pTALEN TET Sar1a (T39N)	This study	N/A
pCMV KDEL-DsRed	This study	N/A
pEYFP-Sec31a	Addgene	66613
AICSDP-25:MAP1LC3B-mEGFP	Addgene	101783
AICSDP-7:Sec61 β–mEGFP	Addgene	87426
PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’)	David Ginsburg	N/A
PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’)	David Ginsburg	N/A
PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’)	David Ginsburg	N/A
Software and algorithms
EasyFRAP	Rapsomaniki et al., 2012	https://doi.org/10.1093/bioinformatics/bts241
IMARIS	Bitplane	N/A
FIJI	Schneider et al., 2012	https://doi.org/10.1038/nmeth.2089

Fluorescence recovery after photobleaching (FRAP) studies were also performed on the Leica SP8 system, using a super continuum white-light laser (65 mW) and an additional 408 nm laser at full power (50 mW). Regions of interest were bleached for 5 frames in a circular pattern, followed by confocal imaging. Data were curated to remove outliers defined as 5 standard deviations from the average intensity at any given timepoint. Analysis was conducted using EasyFRAP. All other image analysis, including co-localization studies and fluorescence intensity measurements, was performed using IMARIS (Bitplane) or FIJI software. For lattice light sheet microscopy studies, beam alignment, dye alignment, and bead alignment were calibrated on the day of each experiment. Point spread functions (PSFs) were generated with numerical apertures of 0.550 and 0.493. Time-lapse studies were conducted in an image format of 1024 × 1024 or 1024 × 512 pixels, stack size of 15–20 μm, and a skewed z-step of 553 nm (deskewed: 300 nm). Each raw time-lapse image series was subsequently deskewed using SlideBook software and processed by constraint iterative deconvolution (3D frequency filter enabled, gaussian noise smoothing at 0.7, and mirrored edge padding (20%) was applied to the x-, y-, and z-planes.

Electron microscopy and immunoblotting studies

For electron microscopy studies, cells were grown on sapphire disks and frozen using a Leica EM ICE high-pressure freezer. Samples were freeze-substituted and embedded through a graded series of Epon EMbed 812, as described previously.⁸³ Micrographs of 80 nm sections were collected on a Phillips CM120 80 kV transmission electron microscope equipped with an AMT Biosprint 12 series digital camera. Immunoblotting studies were conducted as described²² using validated antibodies (see key resources table).

RNA isolation and quantitative PCR

RNA extraction and quantitative PCR (qPCR) analysis were conducted as described previously.⁵⁸ Briefly, RNA was extracted from RPE1 cells using TRIzol reagent according to the manufacturer’s instructions, and used to synthesize cDNA with a Superscript III First Strand RT-PCR kit. RT-qPCR amplifications were performed on the CFX384 Touch Real-time PCR detection system (Bio-Rad). Validated primers were used (see key resources table).

QUANTIFICATION AND STATISTICAL ANALYSIS

For all studies, sample size was determined based on prior experience, and all measurements were randomized and conducted in a blinded manner by two different members of the laboratory to reduce the possibility of incorporating bias. Statistical analysis was conducted using GraphPad Prism software. Linescan analyses and other fluorescence intensity measurements were conducted using Imaris, Nikon Elements, or ImageJ software, following background subtraction. A paired t-test was used to compare two conditions, while multiple conditions were compared using a two-way ANOVA followed by a Tukey’s multiple comparison test. See figure legends for details.

Highlights.

• Loss of Sar1 fails to inhibit the export of multiple secretory cargoes from the ER

• The inner COPII coat complex is indispensable for anterograde protein transport

• COPII coat proteins undergo phase transition in the absence of Sar1