p125A (Sec23ip) couples COPII coat assembly with
donor-acceptor membrane organization to facilitate
tunnel-based traffic

Kimberly R Long, Gazal Singh, Marie Villemeur, Nathalie Brouwers, Vivek

Malhotra, Ishier Raote, Meir Aridor

► To cite this version:

Kimberly R Long, Gazal Singh, Marie Villemeur, Nathalie Brouwers, Vivek Malhotra, et al. p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic. 2025. hal-05395721

HAL Id: hal-05395721

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Preprint submitted on 3 Dec 2025

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p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic.

Kimberly R. Long¹, Gazal Singh¹, Marie Villemeur⁵, Nathalie Brouwers², Vivek

Malhotra²,³,⁴, Ishier Raote²,⁵, and Meir Aridor¹*

¹Department of Cell Biology, University of Pittsburgh School of Medicine, 3500 Terrace

St., Pittsburgh, PA 15261, USA, ²Centre for Genomic Regulation (CRG), The Barcelona

Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain.

³Universitat Pompeu Fabra (UPF), Barcelona, Spain. ⁴ICREA, Pg. Lluis Companys 23,

Barcelona 08010, Spain. ⁵Université Paris Cité, Institut Jacques Monod, CNRS

UMR7592, F-75013 Paris, France

*Corresponding author

Meir Aridor

Email: aridor@pitt.edu

Keywords:

Endoplasmic Reticulum, p125A/Sec23ip, Golgi, COPII, organelle, ERGIC
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## Abstract

COPII-coat proteins play a crucial role in generating small vesicles at endoplasmic reticulum (ER) exit sites (ERES). However, they also assemble at the necks of tunnels and tubules connecting the ER exit site (donor) to the ERGIC/cis-Golgi cisterna (acceptor). How can COPII support these two traffic mechanisms? Through cell-free reconstitutions, we have found that the apposition of donor and acceptor membranes is important for the assembly of the outer layer of the COPII coat (Sec13/31) but had minimal impact on Sar1-induced COPII inner layer (Sec23/24) recruitment. The expression of the adaptor protein p125A, which binds to phosphatidylinositol 4-phosphate (PI4P), Sec31 and Sec23, stabilized the contact between the donor and acceptor membranes and promoted COPII outer layer assembly. A p125A-chimera expressing a Golgi-targeted PI4P-binding domain could also support outer layer assembly. Notably, the C-terminal helical domain of Sec31A, which interacts with p125A, was essential for its assembly at ERES. In cells lacking p125A, the assembly of the COPII outer layer was selectively destabilized. Transcriptome and secretome analyses reveal selective adjustments to extracellular matrix remodeling and collagen secretion, which corresponded with selective inhibition of fibrillar collagen traffic from the ER. Thus, p125A connects the outer COPII layer at ERES with PI4P-rich ERGIC/cis-Golgi membranes, coordinating COPII assembly with tunnel-driven collagen traffic.

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## Introduction

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The activation and recruitment of the small GTP binding protein Sar1 to endoplasmic reticulum exit sites (ERES) initiates a sequential assembly process, where the COPII coat inner layer Sec23/Sec24 is recruited followed by the assembly of outer layer subunits Sec13/Sec31 on a Sar1-Sec23 platform (1). The linking between the two coat layers stimulates GTP hydrolysis allowing for uncoating while facilitating membrane fission(2, 3) (4-6). This assembly process generates small vesicles in vitro from model membranes(7, 8). While small COPII vesicles have been observed in yeast, their visualization in metazoan cells is limited. Additionally, these vesicles are unable to export bulky molecules including extracellular matrix (ECM) proteins, a crucial requirement for animal multicellularity. Recent molecular analyses, combined with nano-resolution microscopy, support the involvement of tunnels or tubules in cargo export from the ER (9, 10). There is evidence suggesting that tunnels form between the donor ERES and the acceptor membranes (ERGIC/cis Golgi) through interactions of the TANGO1 family of ERES proteins with tether and fusion factors (11, 12). In fact, yeast, which also utilizes a similar contact-based traffic mechanism, suggests a simpler form where ER COPII buds transiently connect with Golgi membranes to mediate traffic in a "hug and kiss" type reaction (13).

COPII proteins play a dual role in cargo export at the ER: they can coat small vesicles entirely and also participate in the export of the bulky ECM through tunnels (2). Studies have revealed COPII coats at the necks of tubules emerging from ERES (9, 10). The question arises: how can the vesicle forming COPII coat support tunnel-based traffic? Inter-compartment tunnels could form if a budding carrier fuses to the next compartment before the completion of fission.

However, if and how COPII assembly is coordinated with the recruitment of acceptor membranes remains an open question. p125A (Sec23ip) could play a role in this step of tunnel biogenesis. p125A is a Sec31-binding adaptor and a stoichiometric component of the Sec13/31 complex in the cytosol (14, 15). It interacts with Sec23 upon membrane recruitment, thus physically linking the inner and outer coat layers. This interaction facilitates the coordination of COPII assembly at the ER exit sites (ERES). Moreover,
p125A binds PI4P and supports Sec13/31 assembly at ERES (14, 16). Since PI4P is enriched on acceptor ERGIC and cis Golgi membranes, p125A may be in a unique position, simultaneously interacting with COPII at the ERES and PI4P at downstream compartments. We posit that these activities may coordinate between tunnels/tubules formation and coat-mediated cargo transport. PI4P regulates ERES assembly, adjusts ERES to acute cargo load, and regulates general cargo traffic from the ER (17-19). The utilization of PI4P in ER to Golgi traffic is evolutionarily conserved, regulating COPII vesicle fusion in yeast, which employs transient membrane contacts to support a "hug and kiss" based traffic mechanism (20) (13).

We investigated how COPII assembles at connected ERES-ERGIC-cis Golgi secretion hubs. Our findings demonstrate that p125A plays a role in coupling donor-acceptor membrane contacts with coat assembly, by simultaneously binding Sec31 at the ERES and ERGIC/Golgi membranes. The interaction with p125A facilitates coat outer layer assembly through PI4P binding. Deletion of p125A or removal of its binding site on Sec31 leads to the selective destabilization of COPII outer layer assembly at ERES, resulting in the selective inhibition of tunnel-based traffic from the ER. We propose that p125A serves as a link, coupling acceptor membrane contacts with outer layer coat assembly and GTPase activation, thereby coordinating coat functions with tunnel-based traffic.

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## Results

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### The recruitment of COPII inner and outer layers can be uncoupled.

Multiple redundant mechanisms and proteins maintain ERES-ERGIC function and organization. To overcome this complexity, we designed a 'rundown-type reaction', where cells are permeabilized and then incubated for varying lengths of time before assaying for ERES-ERGIC machinery and function (21). Following permeabilization, cytosolic factors will be gradually released from membranes, diluted, and removed from the cells, leading to a rundown outcome, where dynamic and active processes are reduced progressively. At later times, only the most robust and stable activities will remain, allowing for the dissection of key traffic intermediates. A similar approach was instrumental in analyzing regulated secretion in mast cells (21).

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We adopted a rundown protocol in which permeabilized cells HeLa were pre-incubated at 32°C in the absence of cytosol, followed by the addition of rat liver cytosol with recombinant Sar1-GTP (Sar1<sup>H79G</sup>). The cytosol would provide COPII and regulatory proteins required to reconstitute COPII assembly. Under control conditions, Sar1-GTP induced recruitment of both COPII layers to defined ERES, as expected (Fig. 1). Under the rundown conditions, the recruitment of the coat inner layer (Sec24C) was still effective (Fig. 1B and C-D) but interestingly, the recruitment of the COPII outer layer Sec31 (Fig. 1A, C, H and Fig. 4E) was quantitatively and robustly inhibited whereas the recruitment of Sec24C was largely unaffected (Fig. 1C). We have monitored Sec31 recruitment using two antibodies, a rabbit one that recognizes both human and rat Sec31A (Fig. 1A, 1C, 1H, Fig. 2 and Fig. 4E) and a monoclonal antibody that preferentially recognizes human Sec31A (Fig. 1D) (22). Thus, COPII outer layer proteins are recruited from cytosol but also retained at ERES through cell permeabilization and washes and stabilized by Sar1 activation. In a similar manner the recruitment of Sec13 was inhibited in rundown conditions (Fig. 1E, Fig. S1A). Under the permeabilization conditions, microtubule network disruption leads to changes to the ER network (23). However, ER morphology was similar in control and rundown conditions as marked by an ER localized reporter, a Tac chimera that presents the cytosolic C-terminus of gp25L containing a di-lysine retrieval / retention motif, (Tac-gp25L, Fig. 1E). COPII was recruited by Sar1-GTP from cytosol to ERES showing strong colocalization of Sec31A with Sec16A, that was perturbed when monitored in cells depleted of Sec16A prior to permeabilization and reconstitution of coat assembly (Figs. 1F and S1B). ERES were not disrupted in rundown conditions as monitored by Sec16A localization (Figs 1F and S1B). We further expressed GFP tagged Sec16A to monitor the recruitment of the coat inner layer at ERES. In these cells, recruited Sec24C juxtaposed and overlapped with EGFP-Sec16A at ERES both in control and rundown conditions (Fig. 1G). Overall, the results suggest that under rundown conditions, the COPII inner layer is effectively assembled at ERES while outer layer assembly is perturbed (Fig. 1A-E and G-H and Fig. S1A). We confirmed that Sar1 activation was unaffected under rundown conditions by showing that Sar1-dependent ERES membrane tubulation was unaffected in incubations carried out only in the presence of Sar1-GTP [(3, 24), not shown]. Since the recruitment of the
143 COPII outer layer at ERES was selectively inhibited, it appears that rundown incubations
144 uncouple the two coat layers.

145 **Sec13/31 recruitment to microsomes is inhibited in rundown conditions.**

146 The morphology-based coat assembly assays suggest that the coat inner layer was
effectively assembled at ERES under rundown conditions while the outer later was not –
148 thus Sec23/24 concentration at ERES was independent of Sec13/31 polymerization (Fig.
149 1). We needed an independent robust quantitative assay to further evaluate large
150 populations of ERES and determine the magnitude of the observed rundown effect in a
151 manner that goes beyond morphological analysis. We therefore utilized isolated salt-
washed microsomes to reconstitute membrane binding of COPII proteins following Sar1-
152 activation (25, 26). Incubation of microsomes with cytosol and Sar1-GTP led to the
153 recruitment of both inner and outer COPII layers as monitored by concomitantly following
154 Sec23 and Sec31 on the same western blots (Fig. 2, 2A). The reaction was dependent
155 on the addition of cytosol with Sar1-GTP and membranes as previously described (3, 17,
156 25). These assays started with salt washes to extract peripherally attached proteins from
157 microsomes. Therefore, we could selectively focus on recruitment of cytosolic Sec31A
158 (Fig. 2A). In agreement with our quantitative microscopy-based analysis in
159 permeabilized cells (Fig. 1C), subjecting microsomes to the rundown protocol led to
160 selective inhibition of outer layer recruitment whereas the recruitment of the inner layer
161 was only slightly affected (Fig. 2A-E). The microsome-based assay, which averages
162 millions of cells and ERES, was further used to quantify the overall rundown effect on
163 COPII outer layer recruitment (Figs 1 and 2). For the analysis, we now quantified the
164 ratio between inner and outer layer recruitment on membranes in control and rundown
165 conditions using 22 independent experiments. For each experiment, we defined the ratio
166 between inner (Sec23) and outer layer (re probed for Sec31) per sample/lane on the
167 same western blot, with Sec23 providing an internal control. The results showed an
168 averaged inhibition of 70% in the recruitment of the coat outer layer when compared with
169 inner layer assembly (Fig. 2B) in agreement with morphological analysis (Fig. 1C).
170 Collectively, the results suggest that the rundown incubation led to quantitative
171 uncoupling between COPII inner and outer layers at ERES.
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173 **The rundown response is ATP-independent.**

174 We considered other ERES components that might be affected by the rundown and
175 have a role in this coupling of inner and outer layers. Since the experiment used fresh
cytosol and rundown cell membranes, the component cannot be soluble cytosolic
176 proteins, but it could be membrane-associated peripheral protein. Sec16A is a peripheral
177 membrane protein at ERES that partitions between cytosol and membranes (27, 28) and
178 is known to regulate outer layer membrane recruitment, competing with Sec31 for Sec23
179 binding and inhibiting GTPase activation(29, 30). To test its role in the rundown assay,
we depleted Sec16A using siRNA and assayed whether this rescued Sec13/31
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181 recruitment after rundown. However, we observed no rescue – Sec16A depletion did not
182 relieve the inhibition of Sec13/31 assembly after the rundown (Fig. S1B).
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184 ATP and kinase activities regulate COPII assembly (31) and might contribute to the
185 reduced recruitment of the coat outer layer during the rundown assay. To examine if
kinase activities are modified during rundown incubations, ATP regeneration system and
186 GTP (NTPs) were added or omitted during rundown incubations of washed microsome
187 membranes. Subsequently both NTPs were re-added along with cytosol and Sar1-GTP
188 to support the coat assembly reaction and the recruitment of both coat layers was
monitored. While Sec23 recruitment was slightly enhanced by the presence of NTPs
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190 during the initial rundown incubation, Sec31 binding remained inhibited, suggesting that
191 the phosphorylation status of membrane components is not the cause for the selective
192 inhibition of Sec13/31 recruitment (Fig. 2C).
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194 Another possible option was Phosphatase 2A (PP2A), as it has been shown to function
195 in an early step in ER-exit (32). Treatment of membranes during rundown incubations
with the PP2A inhibitor okadaic acid (OA), inhibited the recruitment of the coat inner
196 layer (Sec23) and did not reverse the inhibition of outer layer assembly (Fig. 2D).
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198 We next tested whether the phospholipid PI4P, which supports COPII assembly on
liposomes and regulates ERES assembly in cells, might rescue the rundown effect (14,
199 17, 18). Addition of short chain PI4P micelles to microsomes during rundown incubations
200 increased the recruitment of Sec23 to membranes in a dose dependent manner (Fig.
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202 2E)(8). However, while these conditions also slightly elevated the recruitment of
203 Sec13/31, the selective inhibition of Sec13/31 assembly in rundown conditions was not
204 reversed (Fig. 2E).

205 The helical C-terminal domain of Sec31 is utilized for assembly at ERES.

206 Having ruled out these additional components or conditions, we considered the
207 possibility that the binding itself between the two coat layers is regulated directly. The
208 links between coat layers are established by the C-terminus of Sec31A, which contains a
209 disordered proline-rich linker and a C-terminal α-solenoid-like helical domain. Within the
210 proline-rich domain is a short segment containing the GAP-active tryptophan and
211 asparagine residues (WN), which binds to an extended interface generated by Sec23
212 and Sar1 (6). These interactions are strengthened by the binding of a triple proline motif
213 (PPP) to the gelsolin domain of Sec23 (33)

214 We analyzed the role of the helical C-terminus in supporting assembly at ERES. We
215 truncated Sec31A to remove the C-terminal helical domain, or the domain along with the
216 preceding proline-rich segment that links to the GAP-active Sar1-Sec23 binding WN
217 domain (Fig. 3B) and verified that the WN-segment binds Sec23 in GST pull down
218 assays (not shown). Because the analyzed constructs preserve the architectural core of
219 Sec31A and can polymerize with ERES-localized endogenous Sec31A, we utilized 3'-
220 UTR targeting siRNA to deplete endogenous Sec31A and replaced it with fluorescently
221 tagged (FP-tagged) rSec31A (rat) proteins (Fig. 3A)(34). Both wildtype and truncated
222 forms of FP-rSec31A were well expressed in control and depleted cells, suggesting that
223 the truncations did not compromise overall protein stability (Fig. 3A and E). For
224 morphological analysis, we permeabilized cells prior to fixation to wash-out
225 overexpressed cytosolic Sec31A and focused on ERES-associated proteins. CFP-
226 rSec31A localized to ERES in control cells, and in Sec31A-depleted cells (Fig 3C). In
227 contrast, both truncated forms of Sec31A showed abnormal localization. In control cells,
228 the FP-rSec31A truncations clustered in few discrete enlarged patches. In Sec31A-
229 depleted cells, both truncated forms largely lost ERES localization and diffused staining
230 was observed at different expression levels within analyzed fields, yet few cells still
231 showed discrete large patch-like morphology (Fig. 3C). Therefore, the truncated Sec31A
232 proteins retain the ability to interact with endogenous Sec31A, most likely through the
233 polymerization core as seen in non-depleted cells, but the C-terminal helical domain of
234 Sec31A was required for proper assembly at ERES. The results suggest that the Sec23-
235 binding WN linker and the polymerization core of Sec31 are insufficient for proper
236 nucleation of the coat outer layer at ERES, the C-terminal helical domain is also
237 required.

238 The C-terminus helical domain of Sec31A binds p125A.

239 Yeast Sec31p utilizes multiple PPP motifs that compete for a single binding site on
240 Sec23 or can also bind adjacent Sar1-Sec23/24 complexes to organize the inner layer in
241 scaffolds (35, 36). These interactions are facilitated by the binding of the C-terminal
242 helical domain onto the central α-solenoid dimerization arms of the Sec13/31 rhomboidal
243 lattice stabilizing cage assembly. Inner- outer layer linking is further supported by
244 multiple charge-based interactions. The reduced number of PPP motifs and reduced
245 dependency of Sec31/13 rhomboidal assembly on the C-terminal α-solenoid in
246 mammals suggest that metazoans and yeast differ in their mode of coat layer linking.
247 Adaptors evolved in mammals to facilitate the linkage between the two coat layers (2,
248 35). Of these, p125A (Sec23ip) is a stoichiometric component of the cytosolic Sec13/31
249 complex that binds Sec23 upon membrane recruitment. We performed pull-down assays
250 using GST-tagged Sec31A domains that affected assembly at ERES and examined
251 interactions with p125A. p125A selectively interacted with a fragment containing the C-
252 terminal α-solenoid-like helical domain, but not with the remaining proline-rich linker (Fig.
253 3D). Importantly, under the analyzed conditions, the C-terminal helical domain-
254 containing fragment depleted both endogenous and expressed GFP-tagged p125A from
255 cell lysates (Fig. 3D). To verify that p125A interacts with the C-terminal helical domain of
256 Sec31A, we also co-expressed mRFP-p125A together with WT CFP-rSec31A or the
257 Sec31A truncated forms in HeLa cells. Over expression of p125A leads to clustering of
258 cargo containing ERES that collect COPII as well as ERGIC membranes and can report
259 on p125A-Sec31 interactions(14, 37). The overexpression of mRFP-p125A effectively
260 collected CFP-rSec31A at enlarged ERES (Fig. 3E). In contrast, the truncated forms of
261 Sec31A did not associate with p125A at ERES but remained cytosolic, confirming that
262 the C-terminal helical domain of Sec31A binds p125A (Fig. 3E).

263 Having identified p125A as an adaptor that could have an important role in this process,
264 we could now propose hypotheses for how COPII-layers are coupled and what changes
265 during the rundown that leads to a loss of outer layer recruitment. In addition to the
266 ability to bind sec31A, p125A is a PI4P-binding adaptor. Interestingly, PI4P is enriched
267 at ERGIC and Golgi membranes. This led us to hypothesize that p125A may bind PI4P
268 that is enriched on acceptor ERGIC / cis Golgi membranes and that these membranes
269 are unavailable for p125A binding in rundown conditions due to a breakdown of ERES-
270 hub like organization (14, 16).

271 **Spatial organization of ERES-hub membranes is disrupted in rundown**
272 **incubations.**

273 ERES are assembled from COPII coated ER membrane buds that are connected to the
274 ERGIC by tunnels. In human cells, these connections appear as single tunnels coated
275 with a COPII collar linked to ERGIC tubules(9). It is possible that connectivity is stable.
276 Alternatively, a dynamic process of budding, fission and fusion is synchronized to
277 generate tunnels (Fig. 7A). In drosophila, ERES-ERGIC tunnels consist of 1-3 fused
278 vesicles supporting transient connectivity(38). To facilitate tunnel-based traffic, delayed
279 fission by coordinating COPII outer layer recruitment with membrane organization, along
280 with increased tethering and fusion may be necessary.

281 We analyzed the localization of exogenously added Sec24C recruited by Sar1-GTP in
282 relation to that of acceptor membranes (ERGIC53, ERGIC) or Giantin (cis Golgi) in
283 control and rundown conditions. Recruited Sec24C partially overlapped both with
284 ERGIC53 membranes at peripheral ERES and with Giantin at the juxtanuclear Golgi
285 region (Fig. 4A and not shown). In contrast, this overlap was lost under rundown
286 conditions. The overall ERGIC53 signal was reduced during the rundown incubation,
287 attesting to a loss of ERGIC membranes from permeabilized cells, as previously
288 described [(39) and see below]. The remaining ERGIC53 signal localized to the ER as
289 we previously reported for these assays(26), making it difficult to spatially define donor
290 and acceptor membranes. We therefore focused on Giantin, which remained intact
under rundown conditions, providing a better view of the spatial organization of the ERES-cis Golgi interface. A dramatic and quantitative loss of overlap between Sec24C and Giantin in the juxtanuclear Golgi region was detected after the rundown (Fig. 4A, quantified in 4B). Thus, contacts between the membranes that make up the ERES hub are destabilized in rundown incubations showing transient connectivity between ERES and its target-acceptor membranes. Consistent with these data, our EM images of ERES after the rundown showed fewer, simpler tubulovesicular clusters, an increase in Sar1-induced tubulation at ERES, and the characteristic decrease in Sec31 immunogold labeling after the rundown (Fig. 1C) (3, 24, 40, 41).

## ERES-ERGIC membranes detachment in microsome-based rundown assays.

If the separation of ERES-ERGIC-cis Golgi (donor-acceptor) membranes uncoupled the two COPII layers, similar membrane separation should also be observed in microsome-based assays. To determine if ER and ERGIC membranes separate during rundown, we subjected control and “rundown” microsomes to differential centrifugation and monitored changes in membrane distribution between a medium speed pellet fraction (MSP) containing heavier membranes, and high-speed pellet fraction (HSP) that contains vesiculo-tubular membranes (42). ER buds (donor membranes) were followed using mSec12 (43, 44) and ERGIC53 was used to mark ERGIC (acceptor membranes). While in control incubations both markers were confined to the MSP, in rundown incubations a fraction of ERGIC53 was found in the HSP. Interestingly, we detected mostly dimers and hexamers of ERGIC53 in this lighter fraction, which had detached from ERES in rundown conditions (Fig. 4C). Oligomeric ERGIC53 resides in a post-ER (ERGIC) compartment, as assembly takes place during ER exit (45). The observed shift of ERGIC53 from the heavy membrane fraction is not a result of budding, as under these incubations cytosolic factors were not provided. These data suggest that the biochemical analysis recapitulated our microscopy-based observations, an initial hub of connected ERES-ERGIC membranes become detached and the ERES separates from the ERGIC during the rundown. Though the microscopy-based morphological changes were more striking than the differential centrifugation outcomes, this is to be expected as there is only partial separation of donor-acceptor membrane using differential centrifugation. Overall, the two assays generated consistent results, suggesting that ERES-hub
322 membranes detach during rundown incubations (Fig. 4A-C). The correlation between
323 loss of membrane apposition and inhibition of coat outer layer assembly supports a
324 model in which COPII assembly is sensitive to donor-acceptor membrane organization
325 of ERES whereby p125A, which binds PI4P, can utilize acceptor membrane binding to
326 support outer layer assembly at ERES (Fig. 7B).

327 We next examined if p125A can reverse the effects on Sec13/31 assembly at ERES in
328 our cell-free rundown assay. p125A was over expressed in HeLa cells that were
329 subsequently subjected to permeabilization and the rundown incubation protocol.
330 Expressed p125A was retained in cells after permeabilization and colocalized with Sar1-
331 recruited Sec24C at ERES both in control and rundown conditions (Fig. 4E). p125A also
332 preserved the apposition between ERES and acceptor cis Golgi membranes under
333 rundown conditions (Compare Fig. 4A to 4D). Importantly, p125A maintained Sec31-
334 Sec24C association at ERES effectively reversing the rundown effect in these
335 incubations (Fig. 4E).

336 Our results suggest that PI4P binding may direct p125A to bind and retain acceptor
337 membranes while supporting COPII assembly. To examine this hypothesis, we utilized a
338 p125A chimera in which the SAM-DDHD PI4P recognition module was replaced with a
339 module containing an oligomerization-defective SAM domain and importantly, a bona
340 fide PI4P-binding domain, the PH domain of FAPP1. The expression of a DDHD-
341 truncated form of p125A in cells inhibits the assembly of endogenous Sec31/13 at ERES
342 yet COPII assembly is restored with the expression of this p125A-FAPP1-PH chimera
343 (14). In rundown assays, expression of the p125A-FAPP1-PH chimera protected
344 membrane contacts at ERES, preserving the close apposition between Giantin and
345 ERES (Fig. 4D). The chimera also preserved association of Sec31 at these sites under
346 rundown conditions (Fig. 4E). Therefore p125A, which binds the C-terminal helical
347 domain of Sec31A, uses PI4P recognition to preserve membrane contacts and COPII
348 assembly under rundown conditions.

349 Tether and fusion proteins including Rab1, p115 or syntaxin18 regulate COPII assembly
350 at ERES (39, 46, 47) and may support membrane apposition. However, the expression
351 of active Rab1-GTP (Rab1a Q70L) gave variable results in the cell free rundown assay.
Although transfected Rab1 was retained in permeabilized cells, it did not robustly reverse the selective recruitment inhibition of the coat outer layer under rundown conditions (Sec13, Fig. S1A). Rab1 and tethers dynamically maintain ERES-ERGIC-cis Golgi contacts thus indirectly regulating coat assembly, whereas p125A allows the coat to detect these membrane contacts. Our cell free assays suggest a simple model in which COPII assembly on ER membranes utilizes p125A to interact in trans with acceptor membranes, effectively coordinating donor-acceptor membrane connectivity with coat assembly (model, Fig. 8B).

p125A is required for the dynamic assembly of the COPII outer layer.

It may be expected that expression of an outer layer associated PI4P-binding adaptor in an invitro assembly assay can protect both donor acceptor membrane contacts and outer layer assembly at ERES. Furthermore, cell-free assays utilize non-physiological stoichiometries of traffic components, and coat dynamics derived from cycles of GTP binding and hydrolysis is negated by the utilization of the Sar1-GTP mutant. We looked for an alternative physiological avenue to examine the role of p125A in directing inner-outer layer coupling. Cell free analyses predict that lack of p125A should selectively destabilize COPII outer layer recruitment at ERES. We utilized CRISPR/Cas9 in U2OS cells to delete p125A, (Fig. 5A). Deleted cells did not upregulate the expression of COPII inner (Sec23) or outer (Sec31A) layers (Fig.5A) making these cells suitable for the analysis of COPII assembly. As we previously reported with acute RNAi-mediated depletion, p125A-KO cells displayed marked effects on Golgi showing massive vesiculation and breakdown of juxtanuclear assembly (Fig. 5B-G and 5I). The effects were mirrored with various cis/medial Golgi markers, including giantin, GRASP65 and gpp130, all presenting vesiculated appearance regardless of overlap (Fig. 5B-G). We used GRASP65 to visualize the reversal of these effects by the expression of WT p125A, but not of a mutant that is deficient in PI4P binding (termed PIX), as we previously quantified in our RNAi-based studies (Fig. 5E) (14). Analysis of KO cells by transmission EM identified Golgi mini stacks, indicating that vesiculated Golgi morphology is a result of unlinking of the Golgi ribbon (Fig. 5C). These changes are typically observed in cells presenting secretion defects and cellular stress. The
morphology of the coat inner layer (Sec24C) was largely unaffected by p125A deletion (Fig. 5F, SR-STED in lower panel and Fig. 5H). Hence p125A is not critical for the recruitment of the coat inner layer and its assembly at ERES. In marked contrast, Sec31A, which displayed a defined punctate appearance of ERES in parental cells, lost this defined localization showing smaller puncta and enhanced diffused appearance likely representing defects in membrane binding and increased cytosolic population (Fig. 5G-I and SR-STED in lower panels). To test for the latter, we subjected parental and p125A-KO cells to a brief permeabilization on ice and monitored the retention of the coat outer layer at ERES. Under these conditions intact Golgi assembly was perturbed over time likely due to the expected disarrayed microtubule organization. In parental cells, Sec31A remained associated with ERES following brief permeabilization while it was mostly lost from p125A-deleted cells within 5 minutes (Fig. 5J and quantified in panels below). The results suggest that p125A stabilizes Sec13/31 assembly at ERES leading to increase in membrane-bound fraction. Overall, the results provide independent validation to cell free analyses. In permeabilized cells, expression of p125A preserves membrane organization and COPII outer layer assembly at ERES. In an orthogonal approach, deleting p125A in cells leads to perturbation of Sec31 assembly and membrane binding. Thus, our model (Fig. 7B) is supported by complimentary, independent experimental approaches.

## 401 Reprograming of ECM Remodeling and Secretion in p125A-Deleted Cells.

Deleting p125A strongly affects COPII outer layer assembly, so we examined how cells adapt to maintain function. We compared the gene expression (transcriptome) of control U2OS and p125A-deleted U2OS cells. While cells of the same group showed little variation (Fig. S2A), p125A-knockout cells had considerable changes (Figs. 6A, S2 and S3), with 1115 genes upregulated and 1252 downregulated (Fig. 6B). Gene Ontology (GO) analysis showed that pathways related to extracellular matrix (ECM) remodeling were most affected, including ECM structure, collagen organization, and metabolism (Fig. 6C). Similarly, many downregulated genes also belonged to ECM-related categories and pathways involved in ECM interactions, remodeling, organization and ones directing collagen biosynthesis, trimerization and fibril assembly were
412 overrepresented (Figs. S3 and S4), indicating a comprehensive and coordinated shift in
413 gene expression to adjust ECM composition and function in response to p125A loss.

414 To explore how cells compensate, we analyzed protein secretion using mass
415 spectrometry (Table 1, Supplemental Tables 2-4). The secretion of many ECM
416 components and modifying enzymes, such as matrix metalloproteinase 9, Matrilin-3, and
417 lysyl oxidase homologs 2 and 4, was inhibited in p125A-knockout cells (Table 1).

418 Consistently, secretion of fibrillar collagens, including COL1A1, was reduced (Table 1).

419 However, a few proteins, like collagen XVI, a non-fibrillar FACIT (fibril-associated
420 collagens with interrupted triple helices) collagen, were hypersecreted (Supplement
421 Table 3). These findings suggest that cells may adjust the ECM composition to
422 compensate for loss of p125A and the potential derived defects in biosynthetic secretion
423 of ECM components including bulky fibrillar cargoes.

424 Spatial sensing by p125A selectively coordinates COPII with collagen traffic.

425 Fibrillar procollagens are too large for COPII vesicles, and both imaging and functional
426 analyses demonstrate that such bulky cargoes are transported via tunnels between
427 ERES and ERGIC membranes (48). We examined if a lack of p125A inhibits procollagen
428 1 secretion from the ER. At steady state, a minimal amount of intracellular fibrillar
429 procollagen (COL1A1) was detected in parental U2OS cells, mainly localizing to the
430 juxtanuclear Golgi region with occasional cells showing more accumulation. In contrast,
431 many of the p125A-KO cells accumulated procollagen in expanded beaded ER-like
432 morphology (Fig. 7A, quantification in lower panel) in agreement with the mass
433 spectrometry analyses (table 1).

434 To examine for potential traffic defects that may account for higher collagen 1 levels in
435 KO cells, we subjected parental and p125A KO U2OS cells to increased temperature
436 (40°C) and ascorbic acid depletion. These treatments interfere with procollagen prolyl
437 hydroxylation and triple helical assembly, leading to accumulation in the ER.

438 Subsequently, cycloheximide and ascorbate were added, and cells were incubated at
439 32°C to initiate traffic. We monitored the clearance of collagen 1 from parental and
440 p125A-KO cells over 90 minutes. Levels of collagen 1 were markedly decreased over
441 the incubation period in parental cells, yet secretion was markedly delayed in KO cells
442 retaining collagen 1 from up to an hour before the beginning of clearance (Fig. 7B,
443 quantification in lower panel). Therefore p125A-KO present a clear defect in collagen 1
444 secretion.

445 To examine traffic from ERES, where p125A-COPII localizes and functions, we analyzed
446 the traffic of collagen 1 to the Golgi complex by quantifying overall co-localization of
447 collagen with a Golgi marker (gpp130) over time using Mander's overlap coefficient. In
448 WT cells procollagen 1 accumulated in the ER began to clear, and increased overlap
449 was observed in the Golgi within 5-10 minutes. In KO cells procollagen 1 mobilization as
450 reflected by Golgi localization was significantly reduced throughout the time course of
451 this analysis (Fig. 7C and quantification in 7D). Therefore, the overall secretion defect of
452 collagen 1 (Fig. 1A-B) is mirrored by a reduced localization of collagen 1 in the Golgi at
453 the initiation of secretion.

454 The defect in collagen secretion may represent a global defect in traffic from the ER.
455 However, secretome analysis does not support a global effect (Table 1 and
456 supplemental tables 2-3). Alternatively, the inability to coordinate COPII layer-linkage
457 with donor-acceptor contacts required for tunnel-based traffic may selectively affect the
458 mobilization of bulky cargo. To explore these possibilities, we utilized the temperature
459 sensitive Vesicular Stomatitis Virus G protein (tsVSV-G), an established reporter for the
460 traffic of small size cargoes for analysis. tsVSV-G accumulates in the ER at a non-
461 permissive temperature (40°C) and effectively mobilizes out of the ER in a synchronous
462 manner when shifted to a permissive temperature (32°C). We followed a GFP-tagged
463 tsVSV-G using live imaging. Upon a temperature shift, the reporter which resided in the
464 reticulum concentrated and mobilized from the ER to the perinuclear region effectively in
465 both WT and KO cells, with an overall similar kinetics (Fig. 7E and supplement movies 1
466 and 2). To quantify ER-Golgi traffic, we monitored the overlap of tsVSV-G with cis Golgi
467 at select time points in fixed cells. Overall, tsVSV-G overlap with cis Golgi was similarly
468 increased in both WT and KO cells suggesting that the traffic defect in p125A deleted
469 cells is selective for collagen (Fig. 7F-G). The results agree with our secretome analysis
470 (tables 1 and S3) and collectively suggest that when spatial sensing of donor-acceptor
471 membrane apposition is perturbed, the coordination between coat assembly and traffic is
472 compromised, leading to selective defects in mobilization of cargo that requires direct
473 non vesicular traffic.

474 Discussion

475
476 The vesicular traffic model offers an elegant solution for preserving compartment
477 identities while facilitating efficient transport of approximately a third of the human
478 proteome between cellular compartments and to various cellular and extracellular
479 destinations. The model has contributed to the identification of vesicle forming coats,
480 crucial components utilized in intracellular traffic(49). However, a vesicle-centric traffic
481 model falls short of addressing the transport of significant cargoes including proteolipid
482 particles and ECM components, particularly fibrillar collagens, that are synthesized in the
483 ER. These cargoes, which include collagens, account for about 20% of the human dry
484 body weigh yet do not conform to the size constraints of COPII vesicles involved in
485 traffic.

486 Accumulating data produced strong evidence for a role of tunnels in supporting general
487 secretion and specifically as an added key mode of traffic from the ER(2), raising a
488 fundamental question: How can vesicle-forming coats function in non-vesicular, tunnel-
489 based traffic. In this work, we address this question by providing evidence that COPII
490 coat assembly is coordinated with donor-acceptor membrane proximity required for
491 connectivity and tunnel-based ER export (Figs 1-4). We provide evidence that p125A
492 functions as a COPII coat adaptor that couples coat-assembly with ERES-ERGIC/cis
493 Golgi organization (Figs 4-5) and is selectively required for tunnel-based traffic (Figs. 6-
494 7).

495 Functional and morphological evidence support direct tunnel-based traffic from the ER,
496 where ERGIC compartments become connected to ERES (2, 9). The morphology of ER
497 exit tunnels suggests that coordination between COPII- vesicle budding-fission and
498 subsequent tethering and fusion generates tunnels composed of chain of 1 to 3
499 connected vesicles (Fig. 8A). This hints at a potential inclination towards traffic route
500 influenced by cargo load. The equilibrium is tilted toward vesicular traffic in drosophila
501 yet prefers tunnels in mammalian cells (9, 38). The formation of tunnels may
502 alternatively be driven by retrograde traffic as implied from the utilization of the NRZ
tether and retrograde fusion proteins in the exit of procollagen from the ER and from the ability to maintain the traffic of small sized cargoes under conditions where COPII is depleted(11, 50). Both models are not mutually exclusive. Regardless of mechanisms that build tunnels, our cell-free assays suggest that building of tunnels between compartments is a dynamic process involving transient connections between donor and acceptor membrane compartments (Figs. 1E and 4). In cell-free conditions, donor-acceptor membrane detachment becomes irreversible due to the imposed (and relatively infinite) dilution of the reactions, leading to an observed *rundown* effect (Figs. 1E and 4). Overall, the assays likely recapitulate a single step in an otherwise dynamic cycles of tethering and fusion that support exit of cargoes from the ER (48, 51).

**p125A connects COPII assembly with the recruitment of ERGIC/cis Golgi to the ERES.**

The mechanisms by which the balance between tunnel or vesicle formation at ERES is executed is unknown. ERES Sec12-cTAGE5-TANGO1-Sec16 fence complexes interact with COPII to facilitate inner layer trapping and Sar1 activation while delaying outer layer assembly (33). The TANGO1 fence further facilitates ERES-ERGIC-cis Golgi membrane recruitment (12). The ability to regulate coat assembly while driving membrane contacts support a role for the TANGO1-cTAGE5 fence in controlling the balance between vesicles and tunnels(38, 52). TANGO1 deletion in drosophila and mammalian cells leads to excessive vesiculation that inhibits overall secretion of a variety of cargoes including that of a small size bulk flow marker that accumulate in the ER at steady state(53). Thus donor-acceptor transient connectivity is critical for effective physiological biosynthetic secretion. However, the deletion of p125A does not inhibit general secretion. Similar outcomes are observed when the p125A binding COPII outer layer is depleted, or in general when COPII functions are compromised leading to selective inhibition of collagen secretion(24, 34, 50, 54-56). A tight nanometer-scale interface between ERES-ERGIC likely allows for processes of coat mediated budding, tether and fusion to proceed even when coat assembly is uncoordinated. In fact, such processes proceed even when COPII levels are severely depleted(50), yet the coordination of coat assembly with tether-mediated contacts and fusion becomes significant for bulky cargoes relying on tunnel-based traffic (Fig. 7).
Our results suggest that ERES-ERGIC-cis Golgi membrane contacts are transmitted to the coat outer layer to coordinate coat assembly that facilitates fission. This is achieved by using a key adaptor that is stoichiometric component of the coat outer layer- p125A. p125A selectively binds to acceptor membranes enriched in PI4P (ERGIC and/or cis-Golgi), while controlling COPII coat assembly at the ERES by linking the two coat layers (Fig. 5 and model in 8B). The cell free coat assembly assays rely on negating Sar1 dynamics locking it in a GTP bound state. The fact that a differential stabilization of the two coat layers is observed in our cell free assays (Figs. 1-2) agrees with previous data showing that expression of a GTPase deficient (Sar1-GTP) in cells does not negate the dynamic exchange of the coat outer layer (Sec13) at ERES (57). Our result show that the linkage between inner and outer layers is governed by added interactions, including the recognition of acceptor membranes by p125A or by a chimera of p125A harboring a bona-fide PI4P-binding domain.

The regulation of inner-outer layer coupling is conserved in evolution. The ERES scaffold protein Sec16p controls inner-outer coat assembly in budding yeast, regulating traffic (29). This regulation is expanded in evolution with added dedicated adaptors that control inner-outer layer coupling to couple physiological needs with coat activities. CREB regulated transcription coactivator 2 (CRTC2) modulates COPII-dependent SREBP1 processing by competing with Sec23A for Sec31A binding (58). COPII adaptors that respond to signaling cues such as elevated Ca²⁺ concentration via proteins like Alg2, or selective lipid signals via p125A, further control coat inner-outer layer coupling. These adaptors interact with different domains on the C-terminus of Sec31, with Alg2 binding the proline rich linker and p125A interacting with the structured-terminal helical domain of Sec31A respectively (Fig. 3D-E). While cryoET reconstruction of COPII assembly on ER membranes in yeast presents a clear inner-outer layer topology(59), both Alg2 and p125A link the mammalian COPII outer layer with membranes and these activities are required for assembly at ERES(60). In the case of p125A lipid-recognition specificizes acceptor membranes, the role of Alg2 in membrane binding is yet undefined. The C-terminal helical domain, which is required for Sec31p functionality and yeast viability(61), directs proper assembly of the outer layer at mammalian ERES (Fig. 3A-C). Over expressed p125A phase separates at ERES and
collects acceptor compartments and this activity requires the Sec23-binding N-terminal IDR and C-terminal PI4P binding(14). We find that expression of p125A or a chimera in which the lipid binding module is replaced with an otherwise Golgi localized PI4P binding domain (Fapp1-PH) rescues the assembly of COPII outer layer in cells (14) and in rundown-based cell free assays (Fig. 4) (15, 62).

p125A is required for collagen traffic.

The destabilization of outer layer assembly observed in p125A deleted cells leads to inhibition of COL1A1 traffic (Figs. 6-7). The significance of this regulation is evident in humans, where a homozygous patient expressing a DDHD-truncated form of p125A, which cannot bind PI4P, exhibits severe phenotypes reminiscent of COPII mutations that reduce neural crest cell migration and inhibit collagen secretion from the ER (63).

Our results suggest that p125A assists in spatial sensing reporting on donor acceptor apposition, which is likely maintained by TANGO1 and cTAGE5 proteins that recruit acceptor membranes to ERES. Yet in our cell free assays, p125A expression preserved membrane apposition in rundown conditions (Fig. 4D). While these results may be derived from p125A over expression, a recent study characterized an interaction between Golgi localized VPS13B and p125A that contribute to the apposition between ERES and cis Golgi membranes. Mutations in VPS13B that affect p125A binding lead to the development of Cohen syndrome, which is characterized, among other, by symptoms indicative of collagen secretion defects. VPS13B binds the N-terminus IDR of p125A whereas its C-terminal PI4P-binding module comprised of a SAM and DDHD domains is independently targeted to Golgi membranes(14). In agreement with our results, the authors find that acute depletion of p125A using RNAi leads to robust inhibition of collagen 1 secretion, and this inhibition is more pronounced than the inhibition of collagen secretion derived from VPS13B depletion(64). The combined results support our model, suggesting that when coat assembly and tunnel formation are not coordinated, collagen does not exit the ER effectively. VPS13B likely acts as a lipid tunnel, helping lipids move between ERES and Golgi membranes. If this process is passive, lipids would flow from Golgi membrane to ER-ERES, reducing membrane tension at ERES to support membrane expansion and tubulation(65). The interactions between p125A-VPS13B are specifically found at perinuclear ERES close to the cis
Golgi. Our recent findings show that perinuclear ERES comprising ~40-50% of the total ERES population are selectively utilized to mobilize collagens, suggesting that localized reduction in membrane tension may assist in collagen secretion from the ER(66).

VPS13B is a minor component in cells, is not detected at peripheral ERES together with p125A and does not compete with Sec31A for p125A binding(64). Its lower expression compared with p125A (and COPII), could mean it fine-tunes lipid composition at select ERES, while p125A facilitates coat assembly across all ERES. Although p125A has no PLA1 enzyme activity when measured in isolation, such activity may be regulated locally at ERES. Whether a p125A enzyme assists in directing lipid flow to and from ERES is a topic for future research.

## COPII, a coat and a collar on transport intermediates.

Our results provide an elegant, simple mechanism by which COPII can be coordinated to support traffic by detecting donor-acceptor membrane contacts and potentially tunnel formation. While it is likely that tunnels can form in the absence of COPII activity(50), the role of COPII in tunnel-based traffic requires further studies. Our work defined a regulated step that depends on membrane apposition: the coupling between the coat inner and outer layers (Fig.7). This coupling controls the rate of GTP hydrolysis by Sar1.

Controlling Sar1-GTPase activation may direct the membrane deforming activities of Sar1-COPII at ERES and participate in coordinating tether and fusion activities (tunnel formation) with Sar1-GTPase-driven fission of traffic intermediates (2, 9, 67). Sar1-GTPase activation may also direct a processive concentration of a select group of cargoes at ERES for traffic (10, 68). Similarly, this regulation may govern the exclusion of ER resident proteins, providing dynamic gating at ERES required to prevent nonspecific content mixing between donor and acceptor membranes(69). The assembly of quasi-rigid hedral cages by Sec13/31 at the donor-acceptor membrane interface may also stabilize membrane connectivity, coupling coat mediated cargo sorting with direct, tunnel-based traffic(70). The utilization of phosphoinositide signals to direct COPII activities may further control other traffic routes from ERES. Future studies should address how PIP signals are utilized to direct COPII to biosynthetic secretion or autophagy/lysosomal degradation pathways.
626 Hub-like clustering of donor-acceptor membranes characterizes many cellular sorting
627 sites that function in the secretory and endocytic pathways where coat activities can be
628 similarly regulated to support vesicular and non-vesicular traffic.

629

630

631

632

633

634

635

636 **Materials and Methods**
637

638

Cells. HeLa or U2OS cells were maintained at sub-confluence in Dulbecco's Modified

639 Eagle's Media (DMEM; HyClone Fisher-Scientific or Lonza. Cat. N°: BE12-604F/U1)

640 supplemented with up to 10 % Fetal Bovine Serum (FBS; Serum Source International,

641 Inc.) and 5% Penicillin-Streptomycin (Cellgro) under standard incubation environment

642 (37°C, 5% CO2). Transfection of HeLa cells was carried out using Lipofectamine 2000

643 reagent (Invitrogen-Life Technologies) according to provided protocol, with optimized

644 DNA concentrations. Transfection of U2OS cells was carried out using X-tremeGENE™

645 ROCHE transfection reagent (Millipore Sigma) according to provided protocol, with

646 optimized DNA concentrations. HeLa derived ER microsomes and rat liver cytosol were

647 prepared as described (42).

648 **Plasmids.** A construct for rat ECFP-RnSec31A expression was kindly provided by Dr. R.

649 Pepperkok (EMBL Heidelberg, Germany). Stop codons at residues Q884 and I959 of

650 ECFP-RnSec31a were generated by site-directed mutagenesis [RnSec31Q884\*_F:

651 5'CCACAGGGGCTGTAGCAACAGCCTTCA3'; RnSec31Q884\*_R:

652 5'TGAAGGCTGTTGCTACAGCCCCTGTGG3'; RnSec31I959\*_F:

653 AAAATTACCAAGAAGCCTTAACCAGACGAGCACCTCATT3'; RnSec31I959\*_R:

654 AATGAGGTGCTCGTCTGGTTAAGGCTTCTTGGTAATTTT3'].

655 The GST-fusion with human HsSec31a1041-1220 was generated by restriction enzyme

656 cloning of HsSec31A PCR fragment into pGEX 4T-1. [HsSec31a_EcoRI_1041_F:

657 5'TCCCCGGAATTCTCAGCTCCAGTACCACTGTCAAGC3'; HsSec31a_Xhol_1220_R:

658 5'CGGCCGCTCGAGTTAGACACCCAGCTTATTGGCCTGGGT3)]. The GST-fusion

659 proteinHsSec31a1041-1113 was generated by site-directed mutagenesis of residue
660 E114 of GST-HsSec31a1041-1220 to a stop codon [HsSec31a_E1114*_F:

661 5'CCTATTCCAGATTAGCACCTCATTCTA3'; HsSec31a_E1114*_F:

662 5'TAGAATGAGGTGCTAATCTGGAATAGG3'].

663 The construct for the expression of Flag-Rab1a-GTP(Q70L) was kindly provided by Dr.

664 Dan Devor (University of Pittsburgh). All constructs expressing FP-tagged p125A
665 proteins were previously described (14). tsVSV-G-GFP was kindly provided by Dr.

666 Jennifer Lippincott-Schwartz (Janelia Farms, HHMI)(71). CD25 (IL2-R alpha, TAC) fused
667 to gp25I C-terminus provided by Dr. Linton M. Traub (University of Pittsburgh). All
668 constructs were verified by sequencing.

669 **siRNA.** HeLa cells were seeded at 3x10⁵ cells/35mm dish. The next morning, cells were
670 transfected with siRNAs to Control (DS NC1; IDT),

671 Sec16L 5' CCAGGUGUUUAAGUUCAUCUACUCC-3' and

672 antisense 3' AAGGUCCACAAAUUCAAGUAGAUGAGG 5',

673 or Sec31 [Sense:5'GGACAGCUUCUCUuccACUCAATA3';

674 Antisense:5'UAUUGAGUGGAAGAGAAGCUGUCCUU3' using Lipofectamine RNAiMAX
675 (Invitrogen-Life Technologies) according to manufacturer's directions. siRNA
676 transfections were repeated at 24 hours post-transfection. At 36 hours, each reaction
677 was split onto glass coverslips in 6-well tissue culture plates. At 60 hours, cells were
678 transfected with pECFP-rSec31a, pECFP-rSec31aQ884*, or pEYFP-Sec31aI959 with
679 Lipofectamine 2000 (Invitrogen-Life Technologies) according to manufacturer's
680 directions. At 72 hours, cells were fixed for direct immunofluorescence or lysed for WB
681 analysis with p125A, Sec31a or p97/VCP ATPase- specific antibodies.

682 **Rundown Biochemical Recruitment.** The recruitment of Sec23 and Sec31 to ER
683 microsomes prepared from HeLa cells was performed as previously described with
684 some alterations (25, 26). Rundown microsomes were pre-incubated in binding reaction
685 mixture lacking NTPs for 15 minutes at 32°C before being added to binding reactions
686 containing NTPs, rat liver cytosol and the indicated concentrations of Sar1 proteins(26).
687 Control microsomes were not pre-incubated in any way. All reactions were incubated at
688 32°C for 15 min and terminated by transfer to ice for 10 min. Membranes were salt
689 washed and collected by centrifugation at 16,000 g for 15 min at 4°C, and membrane
690 pellets were resuspended and resolved on SDS-PAGE gels for Western blot
691 analysis(17, 25, 31). The same blots were probed with anti Sec23 and Sec31 -specific
692 antibodies. All presented assays were repeated at least 3 times with the exception of the
693 PI4P addition (Fig. 2D) which was done twice.

694 **Rundown Morphological Recruitment.** The morphological analysis of coat assembly
695 and tubulation of ERES in semi-intact HeLa cells were performed as previously
696 described with some alterations (17, 25, 42). In brief, HeLa cells were permeabilized with
697 40 µg/ml digitonin in KHM buffer for 5 min on ice, washed with KHM, then rundown
698 reactions were pre-incubated in reaction mixture lacking NTPs for 15 minutes at 32°C
699 before incubation with transport cocktail (26) in the presence of rat liver cytosol at 32°C
700 for 30 minutes to measure coat assembly. Control reactions were not pre-incubated
701 following KHM washes but were directly incubated with transport cocktail. All cells were
702 fixed in 4% formaldehyde in PBS for 15 min at RT for coat assembly analysis with
703 Sec13, Sec24c, Sec31, and Sec31a specific antibodies. All morphological assays were
704 repeated at least 3 times with similar outcomes. Permeabilized cells were labeled and
705 processed for EM as previously described (3, 41). Indirect immunofluorescence was
706 carried out as described previously (26).

707 **Imaging and analysis.** Images (for fixed or live imaging) were acquired on an Olympus
708 Fluoview 1000 confocal system using an inverted microscope (IX-81 Olympus) and
709 60×NA 1.42 PLAPON objective. Images were collected using FV10-ASW V. 02.00.03.10
710 (Olympus Corporation), sized and incorporated in figures without further processing
711 using Adobe Illustrator CC and Canvas X software. STED microscopy was conducted on
712 a Leica SP8 STED 3× microscope (Leica Microsystems, Wetzlar, Germany) equipped
713 with a SuperK EXTREME EXW-12 pulsed white light laser (NKT Photonics Inc.,
714 Portland, OR) as an excitation source, a Katana-08HP pulsed depletion laser as a
715 depletion light source (775 nm; NKT Photonics), and a Leica high-contrast plan
716 apochromat 93× 1.30 numerical aperture (NA) glycerol CS2 objective with motorized
717 correction collar (Leica Microsystems); imaging was controlled by Leica Application Suite
718 X software (LAS X, Leica Microsystems). Immuno gold staining and electron microscopy
719 was conducted as previously described (3, 41). For quantitative analysis, experiments 720 (comprised of randomly collected confocal images of cell fields) were subjected to a 721 constant threshold per experiment. Experiments were analyzed for overall intensities of 722 collagen 1 in cells using Image J (Fiji). Similar collection of confocal images of cell fields 723 with constant threshold application was utilized to define Manders' Overlap Coefficient 724 using the Fiji plugin JACoP. All data were subjected to statistical analysis using two 725 tailed T-test.

726 **Membrane Contact Assay.** HeLa microsomes for rundown reactions were pre- 727 incubated in binding reaction mixture for 15 minutes at 32°C. Control HeLa microsomes 728 were not. Membranes were pelleted at 16,000g for 3 minutes at 4°C [MSP]. Pellets and 729 an aliquot of supernatant was saved for analysis. Half of each supernatant was pelleted 730 at 54,000rpm (179000g) for 20 minutes at 4°C in an ultracentrifuge [HSP]. All pellets and 731 supernatants were resolved on 12% SDS-PAGE gels for Western blot analysis with 732 ERGIC-53 or Sec12 specific antibodies. Western blots were quantified by densitometry 733 using Image J. The experiment was repeated 3 times with similar results.

734 **GST-Pulldowns.** 250µg of GST, GST-HsSec31a1041-1220, or GST-HsSec31a1041- 735 1113 were incubated with Glutathione Sepharose™ 4B (GE Healthcare) beads at 4°C 736 for 1-2h. Unbound GST protein was removed by washing 3 times with 1X Assay Buffer 737 [25mM HEPES, pH7.2; 125mM KOAc; 5mM MgOAc; 2mM EDTA; 2mM EGTA]. HeLa 738 cells expressing pEGFP-p125A, were lysed with 1X Assay Buffer + 1% TritonX-100. Two 739 thirds of a 100mM dish were used for each pulldown reaction. The GST-protein-bound 740 beads were incubated with HeLa lysates at 4°C for 1 hour. Beads were washed 2 times 741 with 1X Assay Buffer and 2 times with 1XPBS. The beads were resuspended in 2X 742 sample buffer and the proteins resolved on SDS-PAGE gels for Western blot analysis 743 with p125A-specific antibodies. PD assays were repeated 3 times with similar results.

744 **Gene-Editing in U2OS cells.** The SEC23IP (p125A) knock out U2OS cell line was 745 created using Crispr-Cas9 genome editing using one gRNA at the 5' end of the SEC23IP 746 gene (sg1: gaagtaactaaatgtctgtg) and one at the 3' end of the gene (sg2: 747 ggagaacgtagtcaattcgg) and a single stranded oligo guiding repair of the site favoring 748 deletion of most of the coding region of the gene (ssODN: 749 aggacagcttccttggtcagacttctattcacacatctgtgacacaattgactacgttctccaagaaaaaccaatagagagttt
taatga). SEC23IP gRNAs were cloned into the BbsI site of px458 (addgene 48138) or
px458-mCherry (the latter having the EGFP gene of px458 replaced by mCherry) after
annealing of the oligo's (sg1: caccgGAAGTAACTAAATGTCTGTG and
aaacCACAGACATTTAGTTACTTCc cloned into px458-mCherry; sg2:
caccgGGAGAACGTAGTCAATTCGG and aaacCCGAATTGACTACGTTCTCCc cloned
into px458). For transfection U2OS cells were trypsinized, counted and transfected while
seeding with 500 ng or each gRNA expressing plasmid and 400 pmol of ssODN. Three
days after transfection transfected cells were FACS sorted as single cells into 96 well
plates for further screening. Single cell clones were expanded and screened by PCR for
presence of the deletion fragment and absence of the wild type fragments using the
following primers: deletion fragment ctgcggagtgatagagtacc and tgctagcaccaaaagcccac;
wild type fragment around sg1 ctgcggagtgatagagtacc and gagggagactacttggctgag; wild
type fragment around sg2 gcctggtagctcctcaggttg and tgctagcaccaaaagcccac. To confirm
the presence of only the deleted gene in the selected clone we performed transcript
analysis by amplifying SEC23IP (p125A) transcripts from cDNA using primers upstream
and downstream of the gRNAs therefore amplifying both wild-type and deleted
transcripts. RNA was isolated using the NZY Total RNA Isolation kit (NZYTech) and
cDNA was prepared using the SuperScript III Reverse Transcriptase (Invitrogen).
Transcripts were amplified using primers ccatggccgagagaaaacctaac and
tcaatgctggggctgttctgg. Sequencing analysis of the obtained transcripts further confirmed
the deletion of the gene in 2 different deletion alleles. Absence of the protein in the
selected line was established by western blotting using a SEC23IP antibodies
(HPA038403, ).

**Sec31 stability in control and KO cells.** For the analysis of Sec31A stability in parental or p125A deleted U2OS cells, cells were washed with ice cold PBS and permeabilized on ice using KHM buffer supplemented with 0.1% saponin (W/V). Cells were washed once with KHM fixed and stained with indicated antibodies.

**Collagen traffic assays.**

Collagen accumulation of and traffic analysis was conducted as previously reported (72).
Briefly cells were washed 3 times with PBS and incubated in DMEM 1%FCS for 3 hrs at
40°C under standard incubation environment (humidified, 5% CO2). Cells were
supplemented with 100µg/ml cycloheximide, 50µM ascorbic acid and 25mM Hepes-KOH (pH 7.2) and incubated for defined periods at 32°C, before transfer to ice, washes (3X ice cold PBS) and fixation for imaging. Cells were fixed and stained for immunofluorescence analysis.

tsVSV-G traffic assays.

Parental and U2OS,Δp125A cells were transfected with a plasmid encoding tsVSV-G-GFP using X-tremeGENE-R according to manufacturer's protocol with optimized DNA concentration. The cells were incubated for 5 hr. at 37°C, DMEM, 10% FCS media was added and the cells were moved to an incubator set at 40°C for an overnight incubation (18 hrs). Media was replaced with Optimem supplemented with 100µg/ml cycloheximide and 25mM HEPES pH-7.4 and cells were incubated at 32°C for indicated times. Cells were fixed and processed for indirect immunofluorescence using indicated antibodies directed at Golgi markers and GFP. For live imaging, tsVSV-G transfections were carried by electroporation using the Neon electroporation system (Invitrogen) according to manufacturer's protocol optimized for U2OS cells. Transfected cells were immediately plated on slides (Mattek corp), incubated at 37°C, for 40 hrs, and transferred to 40°C for an overnight incubation in DMEM, 10%FCS. The cells were washed with ice cold PBS, incubated on a temperature-controlled microscope stage at 32°C in Optimem, 100µg/ml cycloheximide and 25mM HEPES pH-7.4 and imaged continually for indicated times.

**Antibodies:** Polyclonal and monoclonal antibodies against Sec31A, polyclonal anti Sec13, Sec12, Sec23, Sar1 and Sec24C antibodies were kindly provided by Dr. Bill Balch (TSRI, La Jolla CA). Antibody to Sec24C from Invitrogen (#PA5-59101), antibody to Sec31A was from BD Transduction Laboratories (#612351). COPII subunit antibodies were re-verified by functional recruitment assays (using indirect immunofluorescence) and/or by using western blots to follow expression or depletion of target antigens.

Antibodies against p125A were from Bethyl Labs (#MSTP053) and Atlas antibodies (#HPA038403). Antibody against collagen 1 from Abcam (#ab138492). Anti ERGIC53 was from Anzo Life Sciences (ALX-804-602), Antibodies to Giantin and GPP130 were kindly provided by Dr. A. Linstedt (Carnegie Mellon U. Pittsburgh PA), anti-GRASP65 (C-20) from Santa Cruz (# sc-1948). Anti FLAG antibodies (M2, F1804 Sigma),
antibodies to Sec16A, Novus Biologicals (NB100-1799), antibodies to p97/VCP, RDI research diagnostics (RDI PR065278), antibodies to GFP, Polysciences Inc. (24240) and Proteintech (66002-1-ig). Anti-human CD25(IL2-R alpha, TAC) from Ancell immunology research products (174-200).

**Proteins:** Recombinant Sar1-H79G protein was produced and purified as previously described (26, 73). GST and GST tagged hSec31A protein fragments were purified on GS-Sepharose 4B (GE Healthcare Life Science).

**Omics:** Approximately, one million Control U2OS and p125A KO U2OS were seeded in each 60mm tissue culture dish with DMEM, high glucose, GlutaMAX™ Supplement, pyruvate (Gibco; 31966021) containing 10% foetal bovine serum (Gibco; A5256701), 1% penicillin-Streptomycin (Gibco;15140122) and 0.25 mM 2-Phospho-L-ascorbic acid trisodium salt (Sigma; 49752). A day before the experiments, cells were washed three times in PBS and incubated in 5ml of the same medium without serum for 18 hours.

**Transcriptomics:** total RNA isolation was performed using the NucleoSpin RNA Kit (Macherey-Nagel) according to manufacturer's recommendations. Approximately 50 µL RNA eluates were obtained. Concentration and RNA integrity number (RIN) were assessed using DeNovix and RNA ScreenTape assay (Agilent, 5067-5576). RNA library preparation and sequencing were performed by Novogene Co. Ltd. (United Kingdom) using human strand-specific mRNA (WBI-Quantification), directional mRNA library preparation (poly A enrichment) and 2 × 150 bp pair-end configuration (PE) with raw data of 8.0 Gb per sample. The prepared libraries were subsequently pooled for sequencing on the NovaSeq platform (Illumina, San Diego, CA, USA), according to optimal library concentration and data amount. All samples were sequenced in the same batch. During the data quality control process, raw reads of the FASTQ format were processed before clean reads were obtained for downstream analyzes. Novogene conducted quantification of gene expression levels, co-expression analysis, and enrichment analysis based on whole transcriptome RNA-Seq data.

**Proteomics:** after adding protease and phosphatase inhibitors, medium was subjected to centrifugation at 2500g at 4°C for 20 minutes to remove any cells or cellular debris. Medium was then concentrated using centrifugal filter units (Millipore; UFC801024) with a 10 kDa cut off. Approximately, 50µl of concentrated medium was frozen and stored at -
842 80°C till further use. Intracellular proteins were collected using RIPA buffer (SERVA;
843 39244) according to the manufacturer's instructions. Protein concentrations were
844 determined using a Pierce™ BCA Protein Assay Kit (Thermo Scientific; 23225).

845 Mass spectrometry grade chemicals were used, including acetonitrile (ACN), H2O,
846 ammonium bicarbonate (NH4HCO3), formic acid (FA), Tris(2-Carboxyethyl) Phosphine
847 Hydrochloride (TCEP-HCl) and S-Methyl methanethiosulphonate (MMTS)
848 (ThermoFisher Scientific). Sequencing grade Trypsin/Lys C mix was from Promega.

849 Ammonium bicarbonate (NH4HCO3) was from Sigma-Aldrich. For sample preparation, a
850 six-fold volume of cold acetone (–20°C) was added to each sample volume containing
851 10 µg of protein extracts. Vortexed tubes were incubated overnight at -20°C then
852 centrifuged for 10 min at 11,000 rpm at 4°C. Supernatant was removed, then the protein
853 pellets were dissolved in 8M urea, 25 mM NH4HCO3 buffer. Samples were then
854 reduced with 10 mM TCEP-HCl and alkylated with 20 mM MMTS. After a 16-fold dilution
855 in NH4HCO3, samples were digested overnight at 37°C by a mixture of trypsin/Lys C
856 (1/20 Enzyme/Substrate ratio). The digested peptides were loaded and desalted on
857 Evotips Pure, provided by Evosep one (Odense, Denmark) according to manufacturer's
858 procedure.

859 For LC-MS/MS acquisition, samples were analyzed on a timsTOF Pro 2 mass
860 spectrometer (Bruker Daltonics, Bremen, Germany) coupled to an Evosep one system
861 (Evosep, Odense, Denmark) operating with the 30SPD method developed by the
862 manufacturer. Briefly, the method is based on a 44-min gradient and a total cycle time of
863 48 min with a C18 analytical column (0.15 x 150 mm, 1.9µm beads, ref EV-1106)
864 equilibrated at 40°C and operated at a flow rate of 500 nL/min. H2O/0.1 % FA was used
865 as solvent A and ACN/ 0.1 % FA as solvent B. The timsTOF Pro 2 was operated with a
866 DIA-PASEF method comprising 12 pydiAID frames with 3 mass windows per frame
867 resulting in a cycle time of 0.975 seconds as described in Bruker application note LCMS
868 218.

869 Raw MS data files were processed using Spectronaut 18 (Biognosys, Switzerland). Data
870 were searched against the SwissProt Mus Musculus (06 2024, 17219 entries) + Bovine
871 (06 2024, 6046 entries) databases. Contaminating proteins identified only in the Bovine
872 taxonomy were excluded from the final protein list. Specific tryptic cleavages were
873 selected and a maximum of 2 missed cleavages were allowed. The following post-
874 translational modifications were considered for identification: Acetyl (Protein N-term),
875 Oxidation (M), Deamidation (NQ) as variable and MMTS (C) as fixed. The maximum
876 number of variable modifications was set to 3. Identifications were filtered based on a
877 1% precursor and protein Q-value cutoff threshold. The protein LFQ method was set to
878 automatic and the quantity was set at the MS2 level with a cross-run normalization
879 applied. Multivariate statistics on protein measurements were performed using Qlucore
880 Omics Explorer 3.9 (Qlucore AB, Lund, Sweden). A positive threshold value of 1 was set
881 to allow a log2 transformation of abundance data for normalization, i.e. all abundance
882 data values below the threshold are replaced by 1 before transformation. The
883 transformed data were finally used for statistical analysis, i.e. the evaluation of
884 differentially present proteins between two groups using a two-sided Student's t-test. An
885 adjusted p<0.05 was used to filter differential candidates.

886 Author Contributions

887 KRL: conceptualization, experimental work, analysis and editing GS: Experimental work
888 data quantifications and analysis NB: experimental work and analysis, MV: experimental
889 work and analysis, IR, VM: analysis writing and editing. MA: conceptualization,
890 supervision, experimental work, analysis, writing and editing.

891 Competing Interest Statement:

892 The authors declare no competing interests.

893 Acknowledgments

894 We thank Drs W. E. Balch, R. Pepperkok, A. Linstedt, D. C. Devor, L. M. Traub and J.
895 Lippincott-Schwartz for generously providing valuable reagents for the study. We thank
896 the CRG advanced light microscopy unit (ALMU). We thank the Center for Biological
897 Imaging (CBI) at the University of Pittsburgh School of Medicine for help with
898 transmission EM and STED microscopy. We thank the proteomics facility (ProteoSeine)
899 of the Institut Jacques Monod for assistance with the mass spectrometry. The study was
900 initially supported by NIH grant 5R01DK092807 (MA). We further acknowledge the
901 support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the
902 Centro de Excelencia Severo Ochoa (CEX2020-001049,
903
904 MCIN/AEI/10.13039/501100011033) the Generalitat de Catalunya through and the
905 CERCA Programme and to the EMBL partnership. We are grateful to the CRG Core
906 Technologies Programme for their support and assistance in this work. We acknowledge
907 financial support from the following sources: Ministerio de Economía y Competitividad
908 (PID2019-105518GB-100 to VM) and MatSec, from Agence Nationale de la Recherche
909 and AJE202210016216 from Fondation Recherche Medicale (IR). This project has
910 received funding from the European Research Council (ERC) through the European
911 Union's Horizon 2020 research and innovation programme under grant agreement
912 951146-LiquOrg.

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<figure><img src="image_2.png" /><figcaption>Long et al
Fig. 1</figcaption></figure>
buffer lacking nucleotides, cytosol or SAR1 proteins for 15 min at 32°C (rundown) or not (control). Subsequently, a full transport cocktail containing rat liver cytosol (RLC) and SAR1-GTP (hamster SAR1a-H79G, 5µg/220µL) were added as indicated. The reaction was carried out for 15 minutes at 32°C, the cells were washed, fixed and analyzed for Sec31(using a polyclonal anti Sec31A antibody, **A.**) or Sec24C (**B.**) at ERES as we described (3) **C**. The intensity of Sec31A or Sec24C was quantified from 9 fields of cells (20-60 cells per field, each field represented by a circle) derived from 3 to 4 independent experiments. Controls were defined as 100 percent and results analyzed using two-tailed t-test. **D.** Control and rundown reactions were as described in A in the presence of SAR1-GTP and RLC. Fixed cells were processed for IF using anti Sec24C and Sec31A (monoclonal) antibodies as indicated. **E.** Permeabilized cells expressing Tac-gp25I (ER marker) were incubated under control or rundown conditions (as in A) with SAR1-GTP and RLC, fixed and stained for Sec13 (red) and Tac (green). **F.** Analysis of Sec31A recruitment to ERES (marked with anti Sec16A antibody) in permeabilized control or Sec16A-KD cells (staining control) supplemented with Sar1-GTP and RLC as in A was analyzed from 6 fields of cells per condition (circles) from two independent experiments using Pearson Correlation Coefficient (PCC) (shown with two tailed t-test) **G.** Cells expressing GFP-tagged Sec16A were permeabilized and incubated as in A with RLC and Sar1-GTP, fixed and stained for Sec24C (red). Arrows in inserts indicate juxtapose labeling of recruited Sec24C with ERES markers in control and rundown conditions. **H.** Reconstitution of COPII recruitment was conducted as described in A. The cells were fixed and labeled with antibodies to Sec31 (polyclonal) and Sec24C, further fixed and processed for EM (3, 26). Bars are 5µm in A and 200nm in E. Arrows in E point to gold labeling of Sec24C or Sec31 at ERES as indicated.

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<figure><img src="image_3.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>E.</figcaption></figure>

<figure><img src="image_7.png" /></figure>

Fig. 2
Long et al

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**Figure 2.** The recruitment of COPII outer layer to ER membranes is inhibited after a rundown incubation. **A.** Control HeLa microsomes or ones subjected to a pre-incubation rundown protocol as in Fig. 1 were subsequently incubated with SAR1-GTP
(300ng/60µL) and/or RLC as indicated. The microsomes were salt-washed and analyzed for coat recruitment using SDS-PAGE and western blots that were probed, stripped and re-blotted using antibodies against Sec31 and Sec23 as indicated (3, 17, 25). B. The density ratios between recruited Sec31 and Sec23 per lane were quantified for control and rundown incubations carried with Sar1-GTP and RLC, using 22 independent experiments. For each experiment control ratio was set at 100% and experiments were than averaged. The outcome of individual experiments is presented with the overall average, (two-tailed t-test between groups is shown). C. Microsomes were subjected to rundown conditions, where the rundown incubation was done with or without added NTPs (ATP regenerating system and GTP) as indicated. Rundown or control microsomes were subsequently incubated with nucleotides in the presence of RLC and SAR1-GTP as indicated and analyzed for Sec31 and Sec23 recruitment. D. Microsomes were subjected to rundown conditions in the presence or absence of Okadaic Acid (OA, 2.5µM) as indicated. Subsequently, control or rundown microsomes were incubated in the presence of RLC and SAR1-GTP (as in A) and the recruitment of Sec23 and Sec31 was determined. E. Microsomes were subjected to rundown conditions in the presence or absence of increasing concentrations of PI4P micelles (in µM) as indicated (17).

Subsequently, control or rundown microsomes were incubated with SAR1-GTP and RLC as indicated and the recruitment of Sec23 and Sec31 was determined by western blot with specific antibodies as in A.

975
<figure><img src="image_8.png" /></figure>

Fig. 3
Long et al

976

977 **Figure 3. Sec31A requires the C-terminal α-Solenoid-like helical domain for**
978 **effective assembly at ERES and for interactions with p125A. A. Control or Sec31A-**
979 **depleted cells were transfected with resistant clones of YFP-rSec31A (lanes 2 and 6),**
980 CFP-rSec31A (3 and 7) or CFP-rSec31A-884X (4 and 8) and probed with Sec31A
981 antibody to detect endogenous and tagged Sec31A. p97/VCP served as loading control.
982 **B.** Schematic representation of rat Sec31A domains showing wild type (WT), a
983 truncation of the C-terminal α-solenoid-like helical domain (959X rSec31A) and of the C-
984 terminal helical domain and the proline-rich link to the Sec23-binding fragment (884X).
985 The GAP active Sec23-binding fragment is labeled as a red box. **C.** Control or Sec31A
986 depleted cells were transfected with CFP-rSec31A, CFP-rSec31A-959X or CFP-
987 rSec31A-884X and the localization of Sec31A (red) and CFP (green) was defined by IF
988 using specific antibodies. Arrows point to cells showing FP-Sec31A coated ERES or
989 diffuse appearance in cells expressing the indicated FP-Sec31A truncations **D.** GST (1),
990 GST-hSec31a-1041-1220 (2, proline-rich linker and terminal α-solenoid) or GST-
991 hSec31a-1041-1113 (3, proline-rich linker), incubated with HeLa cell-lysates expressing
992 GFP-p125A, collected on GS beads and probed with p125A antibodies. Note
993 aggregation of p125A proteins under these conditions and the binding of both GFP
994 tagged and endogenous p125A by the α-solenoid containing fragment. **E.** Left, the
995 expression of CFP-rSec31A, or truncated forms (959X and 884X) as indicated, probed
996 with polyclonal Sec31 antibody. Right, the cellular localization of co-expressed mRFP-
997 p125A, (red) and CFP-rSec31A WT, 959X and 884X proteins (blue) as indicated
998 (merged images are shown). Arrows point to Sec31-p125A colocalization at ERES, or
999 lack of in cells expressing Sec31A truncations. Bar is 10µm.
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<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

Fig. 4
Long et al

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<caption>**Figure 4.** p125A-mediated PI4P recognition on acceptor membranes preserves ERES-hub architecture and COPII outer layer assembly. **A.** Permeabilized HeLa cells were incubated under control or rundown conditions (as in Fig. 1) as indicated and</caption>
the assembly of Sec24C was induced by the subsequent addition of RLC and SAR1-GTP. COPII assembly at ERES (Sec24C, green) was analyzed with respect to acceptor membranes (Giantin-red). Colocalization (yellow) in merged image is shown. Arrows point to c-localization of Sec24C with Giantin in control but lack of proximity in rundown conditions. B. Average Manders' overlap coefficient between Sec24C and giantin in RLC+SAR1-GTP incubations was defined using 6 cell fields, each represented by a circle (20-40 cells per field) for each condition as recorded in three independent experiments. C. Control or pre-incubated (rundown) microsomes (as in Fig. 2) were directly subjected to Medium Speed centrifugation (MS) and the resulting supernatant were further subjected to High-Speed centrifugation (HS) as indicated (materials and methods). Pellet (P) and supernatant (S) fractions of each step were analyzed for ERES (Sec12) and ERGIC (ERGIC53) membranes. Note the selective mobilization of ERGIC53 dimers (d) and hexamers (h) to the HS pellet under simple rundown incubation conditions. D. HeLa cells were transfected with mRFP-p125A or a mRFP-p125A chimera in which the DDHD domain is replaced with the FAPP1-PH domain(14) as indicated and subjected to the rundown protocol as in A, where the localization of mRFP-p125A proteins and Giantin was defined (note renewed proximity of ERES marked by p125A and Giantin and compare to panel A). Arrows point to reestablished proximity between ERES and cis Golgi in cell expressing p125A constructs as indicated. E. HeLa cells transiently expressing mRFP-p125A or mRFP-p125A chimera, were permeabilized and subjected to control or rundown conditions as in Fig. 1. The cells were subsequently incubated with cytosol and SAR1-GTP. The localization of mRFP-proteins (red) and the recruitment of Sec24C or Sec31 was determined by IF. White arrowheads point to ERES or rescued ERES in rundown conditions that express mRFP-p125A proteins whereas red arrowheads point to neighboring un transfected cells deficient in Sec31 assembly. Labeled nuclei (dapi, blue). Bars in A (5µm), D (20µm) and E (10µm).
<figure><img src="image_19.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>D.</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>F.</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>G.</figcaption></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>STED</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>STED</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>H.</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>I.</figcaption></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

5 Minute
Permeabilization

Fig. 5
Long et al.
Figure 5. The Assembly of COPII outer layer is destabilized in p125A-KO cells. A.

Analysis of p125A, COPII inner layer (Sec23), and outer layer (Sec31A) expression in U2OS and p125A KO cells by western blot as indicated (30 µg of protein loaded per lane, re-probed for VCP/p97 as indicated). B. The localization of Giantin (green) and GPP130 (red) in U2OS or U2OSΔp125A cells as indicated. C. Transmission EM of U2OS or U2OSΔp125A cells showing stacked Golgi elements (arrows). Bar is 1µm. D. The localization of GRASP65 in U2OS or U2OSΔp125A cells. E. U2OS or U2OSΔp125A were transfected with mRFP-p125A or p125A-PIX mutant were analyzed for GRASP65 localization. Arrows point to transfected cells showing variable expression levels. F. The localization of COPII inner layer (Sec24C, green for confocal image, red for STED, lower panel) and cis Golgi (GPP130, red in confocal, green in STED) in U2OS or U2OSΔp125A. Arrows point to Sec24C coated ERES. G. The localization of COPII outer layer (Sec31A, red in confocal, green in STED, lower panel) and cis Golgi (Giantin, green in confocal, red in STED) in U2OS or U2OSΔp125A. Arrows point to Sec31A coated ERES and lack of localization in U2OSΔp125A cells H. The localization of COPII outer (Sec31A, red) and inner (Sec24C, green) layers in U2OS or U2OSΔp125A cells. I. Parental U2OS or U2OSΔp125A cells were stained with Sec31A and GRASP65 antibodies or permeabilized with saponin for 5 min on ice, fixed and stained for GRASP65 and Sec31A. Lower panels, quantification of Sec31A intensity per cell in parental and U2OSΔp125A cells, in start (left) and 5 min. permeabilization on ice (right). Circles represent random cell fields (15-45 cells) derived from two independent experiments, normalized to parental cells (100%) and analyzed using two-tailed t-test. Bars for IF are 10µm.

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<figure><img src="image_31.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_33.png" /></figure>

Fig. 6
Long et al.

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Figure 6. Transcriptomic analysis of U2OSΔp125 and U2OS. A. The co-expression

Venn diagram presents the number of genes that are uniquely expressed within each

group, with the overlapping region showing the number of genes that are co-expressed

in both U2OS and U2OSΔp125. B. Volcano plot of differentially expressed genes

(DEGs) (padj < 0.05 and llog 2 (FoldChange)I > 1) identified between U2OSΔp125 and

U2OS. The x-axis represents the log 2 (FoldChange), y-axis represents statistical

significance for each gene. Green dots denote down-regulated gene expression, red

dots denote up-regulated gene expression in U2OSΔp125. The blue dots denote gene

with unchanged expression. The horizontal dotted line indicates the padj-value cut-off,

while the vertical dotted lines mark the point for no change. C. Dot plot of the Gene

Ontology (GO) enrichment analysis for DEGs. The most significant 30 GO Terms were

selected for display. The x-axis is the ratio of the number of differential genes linked with

the GO Term to the total number of differential genes, and the y-axis is GO Terms. The

size of a point represents the number of genes annotated to a specific GO Term, and the

colour from red to purple represents the significant level of the enrichment. GO pathways

with padj < 0.05 are significant enrichment. (See Supplementary Table 1 for complete

gene lists).

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<figure><img src="image_34.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>D.</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>E.</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>F.</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>G.</figcaption></figure>

<figure><img src="image_40.png" /></figure>

Fig. 7
Long et al.
**Figure 7. Biosynthetic traffic in p125A KO cells.** **A.** The distribution of Sec31A (red) and Collagen 1 (green) in U2OS or U2OSΔp125A as indicated. Lower panel, total intensity of collagen in U2OS or U2OSΔp125A, using 2 independent samples for each quantifying 4 fields of cells per sample (two-tail T-test between groups and the total number of cells quantified per bar are shown with SEM). **B.** U2OS or U2OSΔp125A were incubated at 40°C for 3 hr. in the presence of 1% FCS. Following incubations, ascorbate and cycloheximide were added and the cells were shifted to 32°C for the indicated time to monitor Collagen 1 levels. Lower panel: quantification of remaining collagen 1 levels per cell for each time point as indicated, normalized to time 0. Each circle represents a field with an average of 22 cells. Data are taken from four independent experiments and shown with two tailed T-test between groups and SEM. **C.** U2OS or U2OSΔp125A were incubated as in B for the indicated time points and stained for collagen 1 (green) and GPP130 (red). **D.** Quantification of co-localization (Manders' Overlap Coefficient, MOC) between collagen 1 and GPP130 in U2OS or U2OSΔp125A cells at the indicated time points. Shown are averages from 4 independent experiments where at least 3 fields of cells (~10 cells/field) were quantified per time point per each experiment (with two tail t-test and total number of cells quantified per bar, shown with SEM). **E.** U2OS or U2OSΔp125A as indicated, accumulating transfected tsVSV-G-GFP in the ER at 40°C were shifted to 32°C in the presence of cycloheximide and live-imaging was carried. Indicated time points in minutes, movies in supplement. **F.** U2OS or U2OSΔp125A were shifted to 32°C and incubated as in E, fixed at indicated time points and stained for tsVSV-G-GFP (green) and GPP130 (red). **G.** Quantification of tsVSV-G-GFP and GPP130 overlap (Manders' Overlap Coefficient, MOC) in U2OS or U2OSΔp125A cells at indicated times, averaged from 5 independent experiments where at least 3 fields of transfected cells per experiment were quantified per time point (two tail t-test for each time point shown with SEM). Arrows in panels C, E and F point to the Cis Golgi region. Scale bars are 10μm.

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<figure><img src="image_41.png" /></figure>

B.

<figure><img src="image_42.png" /><figcaption>Fig. 8
Long et al.</figcaption></figure>
1112

1113 **Figure 8. A.** Model describing vesicular vs. tunnel-based traffic. **B.** Model describing

1114 spatial sensing by the COPII coat. 1. The cytosolic COPII inner layer (SAR1-Sec23/24)

1115 is recruited onto donor ERES membranes (left panel). The cytosolic COPII outer layer,

1116 composed of Sec13/31 and its associated adaptor p125A (Sec23ip) is recruited onto

1117 SAR1-Sec23/24 while p125A binds Sec23. Sec13/31 recruitment onto the coat inner

1118 layer is stabilized by selective interactions between p125A and PI4P lipid signals that are

1119 enriched on ERGIC acceptor membranes (right panel).
<table><thead><tr><th>Protein Accession number</th><th>Protein description</th><th>Gene</th><th>U2OS,Δp125 versus U2OS cells</th></tr></thead><tbody><tr><td>P22352</td><td>Glutathione peroxidase 3</td><td>GPX3</td><td>0,02</td></tr><tr><td>P36955</td><td>Pigment epithelium-derived factor</td><td>SERPINF1</td><td>0,04</td></tr><tr><td>P02763</td><td>Alpha-1-acid glycoprotein 1</td><td>ORM1</td><td>0,07</td></tr><tr><td>P14780</td><td>Matrix metalloproteinase-9</td><td>MMP9</td><td>0,19</td></tr><tr><td>P18065</td><td>Insulin-like growth factor-binding protein 2</td><td>IGFBP2</td><td>0,26</td></tr><tr><td>P14543</td><td>Nidogen-1</td><td>NID1</td><td>0,35</td></tr><tr><td>P02771</td><td>Alpha-fetoprotein</td><td>AFP</td><td>0,39</td></tr><tr><td>P19823</td><td>Inter-alpha-trypsin inhibitor heavy chain H2</td><td>ITIH2</td><td>0,40</td></tr><tr><td>Q76M96</td><td>Coiled-coil domain-containing protein 80</td><td>CCDC80</td><td>0,41</td></tr><tr><td>P00749</td><td>Urokinase-type plasminogen activator</td><td>PLAU</td><td>0,42</td></tr><tr><td>Q92876</td><td>Kallikrein-6</td><td>KLK6</td><td>0,42</td></tr><tr><td>Q96S86</td><td>Hyaluronan and proteoglycan link protein 3</td><td>HAPLN3</td><td>0,44</td></tr><tr><td>P01009</td><td>Alpha-1-antitrypsin</td><td>SERPINA1</td><td>0,44</td></tr><tr><td>P02452</td><td>Collagen alpha-1(I) chain</td><td>COL1A1</td><td>0,45</td></tr><tr><td>P02766</td><td>Transthyretin</td><td>TTR</td><td>0,45</td></tr><tr><td>P19883</td><td>Follistatin</td><td>FST</td><td>0,46</td></tr><tr><td>Q14624</td><td>Inter-alpha-trypsin inhibitor heavy chain H4</td><td>ITIH4</td><td>0,47</td></tr><tr><td>Q9GZX9</td><td>Twisted gastrulation protein homolog 1</td><td>TWSG1</td><td>0,48</td></tr><tr><td>O15240</td><td>Neurosecretory protein VGF</td><td>VGF</td><td>0,48</td></tr><tr><td>P00750</td><td>Tissue-type plasminogen activator</td><td>PLAT</td><td>0,49</td></tr><tr><td>Q14767</td><td>Latent-transforming growth factor beta-binding protein 2</td><td>LTBP2</td><td>0,50</td></tr><tr><td>P35555</td><td>Fibrillin-1</td><td>FBN1</td><td>0,50</td></tr><tr><td>Q8IUX7</td><td>Adipocyte enhancer-binding protein 1</td><td>AEBP1</td><td>0,50</td></tr><tr><td>P07996</td><td>Thrombospondin-1</td><td>THBS1</td><td>0,50</td></tr><tr><td>P12109</td><td>Collagen alpha-1(VI) chain</td><td>COL6A1</td><td>0,51</td></tr><tr><td>P01024</td><td>Complement C3</td><td>C3</td><td>0,51</td></tr><tr><td>Q96JB6</td><td>Lysyl oxidase homolog 4</td><td>LOXL4</td><td>0,53</td></tr><tr><td>P24593</td><td>Insulin-like growth factor-binding protein 5</td><td>IGFBP5</td><td>0,54</td></tr><tr><td>Q9UNW1</td><td>Multiple inositol polyphosphate phosphatase 1</td><td>MINPP1</td><td>0,54</td></tr><tr><td>P02765</td><td>Alpha-2-HS-glycoprotein</td><td>AHSG</td><td>0,56</td></tr><tr><td>P29279</td><td>CCN family member 2</td><td>CCN2</td><td>0,56</td></tr><tr><td>P12110</td><td>Collagen alpha-2(VI) chain</td><td>COL6A2</td><td>0,57</td></tr><tr><td>P20908</td><td>Collagen alpha-1(V) chain</td><td>COL5A1</td><td>0,57</td></tr><tr><td>Q9UKZ9</td><td>Procollagen C-endopeptidase enhancer 2</td><td>PCOLCE2</td><td>0,58</td></tr><tr><td>Q6UXH8</td><td>Collagen and calcium-binding EGF domain-containing protein 1</td><td>CCBE1</td><td>0,58</td></tr><tr><td>Q9Y4K0</td><td>Lysyl oxidase homolog 2</td><td>LOXL2</td><td>0,58</td></tr><tr><td>O15232</td><td>Matrilin-3</td><td>MATN3</td><td>0,58</td></tr><tr><td>P05121</td><td>Plasminogen activator inhibitor 1</td><td>SERPINE1</td><td>0,60</td></tr><tr><td>P24821</td><td>Tenascin</td><td>TNC</td><td>0,60</td></tr><tr><td>P55001</td><td>Microfibrillar-associated protein 2</td><td>MFAP2</td><td>0,61</td></tr><tr><td>P01011</td><td>Alpha-1-antichymotrypsin</td><td>SERPINA3</td><td>0,61</td></tr><tr><td>Q14766</td><td>Latent-transforming growth factor beta-binding protein 1</td><td>LTBP1</td><td>0,62</td></tr><tr><td>Q6UXH1</td><td>Protein disulfide isomerase CRELD2</td><td>CRELD2</td><td>0,63</td></tr><tr><td>P13674</td><td>Prolyl 4-hydroxylase subunit alpha-1</td><td>P4HA1</td><td>0,64</td></tr><tr><td>Q15113</td><td>Procollagen C-endopeptidase enhancer 1</td><td>PCOLCE</td><td>0,65</td></tr><tr><td>P98160</td><td>Basement membrane-specific heparan sulfate proteoglycan core protein</td><td>HSPG2</td><td>0,66</td></tr></tbody></table>

Tab. 1
Long et al.

1120

1121

1122 Table 1. List of proteins identified as hypo-secreted in U2OSΔp125 compared to
1123 U2OS. Secreted and intracellular proteins were quantified by LC-MS/MS in U2OSΔp125

1124 and U2OS cells. Within each group, the proportion of the secreted protein was

1125 determined. Then, each value for the U2OSΔp125 was compared to the control U2OS.

1126 46 proteins were finally identified as less secreted with a threshold set at 0.67. (See

1127 Supplementary Table 4 for complete proteins lists detected in lysate and secretome).

1128

1129
A.

<figure><img src="image_43.png" /></figure>

B.

<figure><img src="image_44.png" /></figure>

Fig. S1
Long et al

1130

1131 **Supplement Figure 1.** Active Rab1 or Sec16A depletion do not rescue the linkage

1132 between COPII inner and outer layers. **A.** Cells were transfected with construct

1133 expressing Flag-Rab1a-GTP(Q70L), permeabilized and subjected to rundown and
1134 COPII recruitment as in Fig. 1, monitoring Sec13 (red) and Rab1-GTP (anti Flag, green).

1135 Note that Rab1a-GTP is retained in cells and occasionally preserves Sec13 at the sites.

1136 B. Sec16A was depleted using siRNA and analyzed by western blot with Sec31A
1137 serving as a control as indicated (triplicates of control or KD samples are shown in the
1138 left panel). Depleted cells were permeabilized and subjected to rundown pre-incubation.

1139 Subsequently, control or Sec16A depleted cells were supplemented with RLC and
1140 SAR1-GTP (as in Fig.1). The recruitment of the coat outer layer (Sec31A, monoclonal
1141 antibody) and Sec16A localization was determined by IF. Bars are 5 µm.
A.

<figure><img src="image_45.png" /></figure>

B.

<figure><img src="image_46.png" /></figure>

Sup. Fig. 2
Long et al.

1142

1143 Supplement Figure 2. A. Heatmap of Pearson correlation coefficient matrix. Correlation

1144 coefficient between the samples and conditions plotted as heat maps. The higher the
1145 Pearson correlation coefficient (R²) of the sample is, the closer the expression pattern.

1146 The bar on the right of the figure indicates the color legend of R² calculated for each pair

1147 of samples in the matrix. The closer the correlation coefficient is to 1, the higher

1148 similarity between samples. Encode suggests that R² should be greater than 0.92

1149 (ENCODE Project Consortium, 2004). **B. Heatmap of differentially expressed genes**

1150 (padj < 0.05 and llog2 (FoldChange)I > 1) identified between the U2OSΔp125 and

1151 U2OS. Genes or samples with similar expression patterns in the heat map cluster

1152 together. The color in each grid reflects not the gene expression value, but the value

1153 obtained after homogenizing the expression data rows (generally between -2 and 2).

1154 Therefore, the colors in the heat map can only be compared vertically (the expression of

1155 the same gene in different samples), but not horizontally (the same sample). There are

1156 not only inter-group clustering, but also inter-sample clustering.
<figure><img src="image_47.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_48.png" /></figure>

Sup. Fig. 3
Long et al.

1157

1158 **Supplement Figure 3.** Dot plot of the Gene Ontology (GO) enrichment analysis for

1159 down regulated genes (A.) and up regulated genes (B.). The most significant 30 GO
1160 Terms were selected for display. The x-axis is the ratio of the number of differential genes linked with the GO Term to the total number of differential genes, and the y-axis is 1161 GO Term. The size of a point represents the number of genes annotated to a specific 1162 GO Term, and the color from red to purple represents the significant level of the 1163 enrichment. GO pathways with padj < 0.05 are significant enrichment. (See 1164 Supplementary Table 1 for complete genes lists). 1165
<figure><img src="image_49.png" /><figcaption>Sup. Fig. 4
Long et al.</figcaption></figure>

1166

1167 Supplement Figure 4. Reactome enrichment analysis. The most significant 20

1168 Reactome pathways were selected for display. The x-axis is the ratio of the number of
1169 differential genes linked with the Reactome pathway to the total number of differential
genes, and the y-axis is Reactome Pathways as listed. The size of a point represents
1170 the number of genes annotated to a specific Reactome pathway, and the color from red
1171 to purple represents the significant size of the enrichment. (See Supplementary Table 1
1172 for complete genes lists).
1173

1174

1175

1176 Supplement table1. Transcriptomics Data (Excel attached).

1177 1. The full list of differentially expressed genes identified between U2OSΔp125 and
1178 U2OS. 2. List of significantly up regulated expressed genes identified in U2OSΔp125
1179 compared to U2OS. 3. List of significantly down regulated expressed genes identified in
1180 U2OSΔp125 compared to U2OS group.4. List of differentially expressed genes involved
1181 in the most significant 30 GO Terms. 5. List of down regulated expressed genes in
1182 U2OSΔp125 involved in the most significant 30 GO Terms. 6. List of up regulated
1183 expressed genes in the U2OSΔp125 involved in the most significant 30 GO Terms. 7.
1184 List of differentially expressed genes involved in the most significant 20 Reactome
1185 pathways.
<table><thead><tr><th>Protein Accession number</th><th>Protein description</th><th>Gene</th><th>U2OS,Δp125 versus U2OS cells</th></tr></thead><tbody><tr><td>Q8IXL7</td><td>Methionine-R-sulfoxide reductase B3</td><td>MSRB3</td><td>0,67</td></tr><tr><td>P02649</td><td>Apolipoprotein E</td><td>APOE</td><td>0,68</td></tr><tr><td>P12111</td><td>Collagen alpha-3(VI) chain</td><td>COL6A3</td><td>0,69</td></tr><tr><td>P12107</td><td>Collagen alpha-1(XI) chain</td><td>COL11A1</td><td>0,69</td></tr><tr><td>Q9Y680</td><td>Peptidyl-prolyl cis-trans isomerase FKBP7</td><td>FKBP7</td><td>0,70</td></tr><tr><td>Q13421</td><td>Mesothelin</td><td>MSLN</td><td>0,72</td></tr><tr><td>P02768</td><td>Albumin</td><td>ALB</td><td>0,72</td></tr><tr><td>P06396</td><td>Gelsolin</td><td>GSN</td><td>0,72</td></tr><tr><td>P30101</td><td>Protein disulfide-isomerase A3</td><td>PDIA3</td><td>0,74</td></tr><tr><td>Q16270</td><td>Insulin-like growth factor-binding protein 7</td><td>IGFBP7</td><td>0,76</td></tr><tr><td>Q13253</td><td>Noggin</td><td>NOG</td><td>0,76</td></tr><tr><td>O00469</td><td>Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2</td><td>PLOD2</td><td>0,76</td></tr><tr><td>P13987</td><td>CD59 glycoprotein</td><td>CD59</td><td>0,76</td></tr><tr><td>Q9U42</td><td>Carboxypeptidase A4</td><td>CPA4</td><td>0,77</td></tr><tr><td>O95302</td><td>Peptidyl-prolyl cis-trans isomerase FKBP9</td><td>FKBP9</td><td>0,77</td></tr><tr><td>O15230</td><td>Laminin subunit alpha-5</td><td>LAMA5</td><td>0,77</td></tr><tr><td>Q99985</td><td>Semaphorin-3C</td><td>SEMA3C</td><td>0,78</td></tr><tr><td>Q86UN3</td><td>Reticulon-4 receptor-like 2</td><td>RTN4RL2</td><td>0,82</td></tr><tr><td>Q8WWX9</td><td>Selenoprotein M</td><td>SELENOM</td><td>0,82</td></tr><tr><td>O75326</td><td>Semaphorin-7A</td><td>SEMA7A</td><td>0,83</td></tr><tr><td>P07711</td><td>Procathepsin L</td><td>CTSL</td><td>0,84</td></tr><tr><td>P55290</td><td>Cadherin-13</td><td>CDH13</td><td>0,85</td></tr><tr><td>P05155</td><td>Plasma protease C1 inhibitor</td><td>SERPING1</td><td>0,85</td></tr><tr><td>P23142</td><td>Fibulin-1</td><td>FBLN1</td><td>0,86</td></tr><tr><td>Q8NB37</td><td>Glutamine amidotransferase-like class 1 domain-containing protein 1</td><td>GATD1</td><td>0,86</td></tr><tr><td>Q7Z3B1</td><td>Neuronal growth regulator 1</td><td>NEGR1</td><td>0,88</td></tr><tr><td>Q13361</td><td>Microfibrillar-associated protein 5</td><td>MFAP5</td><td>0,90</td></tr><tr><td>Q16610</td><td>Extracellular matrix protein 1</td><td>ECM1</td><td>0,91</td></tr><tr><td>Q9GZM7</td><td>Tubulointerstitial nephritis antigen-like</td><td>TINAGL1</td><td>0,91</td></tr><tr><td>Q8WTU2</td><td>Scavenger receptor cysteine-rich domain-containing group B protein</td><td>SSC4D</td><td>0,93</td></tr><tr><td>Q08397</td><td>Lysyl oxidase homolog 1</td><td>LOXL1</td><td>0,93</td></tr><tr><td>Q9NZP8</td><td>Complement C1r subcomponent-like protein</td><td>C1RL</td><td>0,93</td></tr><tr><td>P15848</td><td>Arylsulfatase B</td><td>ARSB</td><td>0,95</td></tr><tr><td>P10909</td><td>Clusterin</td><td>CLU</td><td>0,96</td></tr><tr><td>O00584</td><td>Ribonuclease T2</td><td>RNASET2</td><td>0,97</td></tr><tr><td>Q9H4F8</td><td>SPARC-related modular calcium-binding protein 1</td><td>SMOC1</td><td>0,97</td></tr><tr><td>P28300</td><td>Protein-lysine 6-oxidase</td><td>LOX</td><td>1,00</td></tr><tr><td>P01019</td><td>Angiotensinogen</td><td>AGT</td><td>1,02</td></tr><tr><td>P20742</td><td>Pregnancy zone protein</td><td>PZP</td><td>1,02</td></tr><tr><td>P22304</td><td>Iduronate 2-sulfatase</td><td>IDS</td><td>1,02</td></tr><tr><td>P16870</td><td>Carboxypeptidase E</td><td>CPE</td><td>1,05</td></tr><tr><td>P15289</td><td>Arylsulfatase A</td><td>ARSA</td><td>1,06</td></tr><tr><td>P38571</td><td>Lysosomal acid lipase/cholesteryl ester hydrolase</td><td>LIPA</td><td>1,11</td></tr><tr><td>P08253</td><td>72 kDa type IV collagenase</td><td>MMP2</td><td>1,15</td></tr><tr><td>P50454</td><td>Serpin H1</td><td>SERPINH1</td><td>1,16</td></tr><tr><td>P10619</td><td>Lysosomal protective protein</td><td>CTSA</td><td>1,18</td></tr><tr><td>O43852</td><td>Calumenin</td><td>CALU</td><td>1,19</td></tr><tr><td>Q13753</td><td>Laminin subunit gamma-2</td><td>LAMC2</td><td>1,27</td></tr><tr><td>P04066</td><td>Tissue alpha-L-fucosidase</td><td>FUCA1</td><td>1,28</td></tr><tr><td>Q99538</td><td>Legumain</td><td>LGMN</td><td>1,35</td></tr><tr><td>P13284</td><td>Gamma-interferon-inducible lysosomal thiol reductase</td><td>IFI30</td><td>1,37</td></tr><tr><td>Q08431</td><td>Lactadherin</td><td>MFGE8</td><td>1,43</td></tr><tr><td>Q92520</td><td>Protein FAM3C</td><td>FAM3C</td><td>1,45</td></tr></tbody></table>

Sup Tab. 2
Long et al.

1186

1187

1188
1189 **Supplement Table 2.** List of proteins with unchanged secretion in U2OSΔp125

1190 compared to U2OS. Secreted and intracellular proteins were quantified by LC-MS/MS in

1191 U2OSΔp125 and U2OS. Within each group, the proportion of the secreted protein

1192 detected was determined. Then, each value for U2OSΔp125 was compared to U2OS.

1193 54 proteins were identified with unchanged secretion (threshold between 0.67 and 1.50).

1194 (See Supplementary Table 4 for complete proteins lists detected in lysate and

1195 secretome).

1196
<table><thead><tr><th>Protein Accessions number</th><th>Protein description</th><th>Gene</th><th>U2OS,Δp125 versus U2OS cells</th></tr></thead><tbody><tr><td>P0C0L4</td><td>Complement C4-A</td><td>C4A</td><td>1,52</td></tr><tr><td>P54803</td><td>Galactocerebrosidase</td><td>GALC</td><td>1,55</td></tr><tr><td>O43854</td><td>EGF-like repeat and discoidin Hike domain-containing protein 3</td><td>EDIL3</td><td>1,60</td></tr><tr><td>Q92485</td><td>Acid sphingomyelinase-like phosphodiesterase 3b</td><td>SMPDL3B</td><td>1,61</td></tr><tr><td>Q9Y2I2</td><td>Netrin-G1</td><td>NTNG1</td><td>1,66</td></tr><tr><td>O94919</td><td>Endonuclease domain-containing 1 protein</td><td>ENDOD1</td><td>1,68</td></tr><tr><td>P01023</td><td>Alpha-2-macroglobulin</td><td>A2M</td><td>1,70</td></tr><tr><td>P04156</td><td>Major prion protein</td><td>PRNP</td><td>1,72</td></tr><tr><td>P08493</td><td>Matrix Gla protein</td><td>MGP</td><td>2,04</td></tr><tr><td>O60568</td><td>Multifunctional procollagen lysine hydroxylase and glycosyltransferase LH3</td><td>PLOD3</td><td>2,06</td></tr><tr><td>O15041</td><td>Semaphorin-3E</td><td>SEMA3E</td><td>2,45</td></tr><tr><td>P17405</td><td>Sphingomyelin phosphodiesterase</td><td>SMPD1</td><td>2,49</td></tr><tr><td>Q07092</td><td>Collagen alpha-1(XVI) chain</td><td>COL16A1</td><td>3,26</td></tr><tr><td>P02753</td><td>Retinol-binding protein 4</td><td>RBP4</td><td>5,78</td></tr><tr><td>Q96S16</td><td>JmjC domain-containing protein 8</td><td>JMJD8</td><td>7,72</td></tr></tbody></table>

Sup Tab. 3
Long et al.

1197

1198

1199
1200 **Supplement Table 3.**

1201 **List of proteins identified as hypersecreted in U2OSΔp125 compared to U2OS.**

1202 Secreted and intracellular proteins were quantified by LC-MS/MS in U2OSΔp125 and U2OS. Within each group, the proportion of the secreted protein detected was determined. Then, each value for U2OSΔp125 was compared to U2OS. 15 proteins were finally identified as hypersecreted with a threshold set at 1.50. (See Supplementary Table 4 for complete proteins lists detected in lysate and secretome).

1207

1208 **Supplement Table 4.** (Excel attached).

1209 Proteomics data. 1, The complete list of intracellular proteins identified and quantified in U2OSΔp125 and U2OS. Fold changes were calculated. P values were calculated using Student's t test. 2. The complete list of secreted proteins identified and quantified in U2OSΔp125 and U2OS. Fold changes were calculated. P values were calculated using Student's t test.

1214

1215

1216 **Supplement Movie 1.** U2OS cells expressing tsVSV-G-GFP were transferred from 40°C to 32°C with added cycloheximide (materials and methods) and directly imaged at mid cell at a rate of 7.05sec/frame, recording time is 20 min.

1219

1220 **Supplement Movie 2.** U2OSΔp125A cells expressing tsVSV-G-GFP were transferred from 40°C to 32°C with added cycloheximide (materials and methods) and directly imaged at mid cell at a rate of 7.05sec/frame, recording time is 20 min.

1223

1224
1225

1226 **References.**

1227

1228 1. Aridor M. COPII gets in shape: Lessons derived from morphological aspects of early secretion. Traffic. 2018;19(11):823-39.

1230 2. Aridor M. A tango for coats and membranes: New insights into ER-to-Golgi traffic. Cell Rep. 2022;38(3):110258.

1232 3. Bielli A, Haney CJ, Gabreski G, Watkins SC, Bannykh SI, Aridor M. Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission. J Cell Biol. 2005;171(6):919-24.

1235 4. Stagg SM, LaPointe P, Razvi A, Gurkan C, Potter CS, Carragher B, et al. Structural basis for cargo regulation of COPII coat assembly. Cell. 2008;134(3):474-84.

1237 5. Fath S, Mancias JD, Bi X, Goldberg J. Structure and organization of coat proteins in the COPII cage. Cell. 2007;129(7):1325-36.

1239 6. Bi X, Mancias JD, Goldberg J. Insights into COPII coat nucleation from the structure of Sec23.Sar1 complexed with the active fragment of Sec31. Dev Cell. 2007;13(5):635-45.

1242 7. Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, et al. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994;77(6):895-907.

1245 8. Matsuoka K, Orci L, Amherdt M, Bednarek SY, Hamamoto S, Schekman R, et al. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell. 1998;93(2):263-75.

1248 9. Weigel AV, Chang CL, Shtengel G, Xu CS, Hoffman DP, Freeman M, et al. ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER. Cell. 2021;184(9):2412-29 e16.

1251 10. Shomron O, Nevo-Yassaf I, Aviad T, Yaffe Y, Zahavi EE, Dukhovny A, et al. COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers. J Cell Biol. 2021;220(6).
11. Raote I, Ortega-Bellido M, Santos AJ, Foresti O, Zhang C, Garcia-Parajo MF, et al. TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes. Elife. 2018;7.

12. Santos AJ, Raote I, Scarpa M, Brouwers N, Malhotra V. TANGO1 recruits ERGIC membranes to the endoplasmic reticulum for procollagen export. Elife. 2015;4.

13. Kurokawa K, Okamoto M, Nakano A. Contact of cis-Golgi with ER exit sites executes cargo capture and delivery from the ER. Nat Commun. 2014;5:3653.

14. Klinkenberg D, Long KR, Shome K, Watkins SC, Aridor M. A cascade of ER exit site assembly that is regulated by p125A and lipid signals. J Cell Sci. 2014;127(Pt 8):1765-78.

15. Ong YS, Tang BL, Loo LS, Hong W. p125A exists as part of the mammalian Sec13/Sec31 COPII subcomplex to facilitate ER-Golgi transport. J Cell Biol. 2010;190(3):331-45.

16. Inoue H, Baba T, Sato S, Ohtsuki R, Takemori A, Watanabe T, et al. Roles of SAM and DDHD domains in mammalian intracellular phospholipase A1 KIAA0725p. Biochim Biophys Acta. 2012;1823(4):930-9.

17. Blumental-Perry A, Haney CJ, Weixel KM, Watkins SC, Weisz OA, Aridor M. Phosphatidylinositol 4-phosphate formation at ER exit sites regulates ER export. Dev Cell. 2006;11(5):671-82.

18. Farhan H, Weiss M, Tani K, Kaufman RJ, Hauri HP. Adaptation of endoplasmic reticulum exit sites to acute and chronic increases in cargo load. EMBO J. 2008;27(15):2043-54.

19. Bryant KL, Baird B, Holowka D. A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIalpha is important for protein trafficking from the endoplasmic reticulum to the plasma membrane. BMC Cell Biol. 2015;16:5.

20. Lorente-Rodriguez A, Barlowe C. Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments. Mol Biol Cell. 2011;22(2):216-29.

21. Pinxteren JA, O'Sullivan AJ, Larbi KY, Tatham PE, Gomperts BD. Thirty years of stimulus-secretion coupling: from Ca(2+) toGTP in the regulation of exocytosis. Biochimie. 2000;82(4):385-93.
1285 22. Biazik J, Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL. Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy. 2015;11(3):439-51.

1288 23. Plutner H, Davidson HW, Saraste J, Balch WE. Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment. J Cell Biol. 1992;119(5):1097-116.

1291 24. Long KR, Yamamoto Y, Baker AL, Watkins SC, Coyne CB, Conway JF, et al. Sar1 assembly regulates membrane constriction and ER export. J Cell Biol. 2010;190(1):115-28.

1294 25. Pathre P, Shome K, Blumental-Perry A, Bielli A, Haney JH, Alber S, et al. Activation of Phospholipase D by the Small GTPase Sar1 is Required to support COPII Assembly and ER Export. EMBO J. 2003;22:4059-68.

1297 26. Aridor M, Bannykh SI, Rowe T, Balch WE. Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport. J Cell Biol. 1995;131(4):875-93.

1300 27. Maeda M, Katada T, Saito K. TANGO1 recruits Sec16 to coordinately organize ER exit sites for efficient secretion. J Cell Biol. 2017;216(6):1731-43.

1302 28. Iinuma T, Shiga A, Nakamoto K, O'Brien MB, Aridor M, Arimitsu N, et al. Mammalian Sec16/p250 plays a role in membrane traffic from the endoplasmic reticulum. J Biol Chem. 2007;282(24):17632-9.

1305 29. Kung LF, Pagant S, Futai E, D'Arcangelo JG, Buchanan R, Dittmar JC, et al. Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat. EMBO J. 2012;31(4):1014-27.

1308 30. Yorimitsu T, Sato K. Insights into structural and regulatory roles of Sec16 in COPII vesicle formation at ER exit sites. Mol Biol Cell. 2012;23(15):2930-42.

1310 31. Aridor M, Balch WE. Kinase signaling initiates coat complex II (COPII) recruitment and export from the mammalian endoplasmic reticulum. J Biol Chem. 2000;275(46):35673-6.

1313 32. Davidson HW, McGowan CH, Balch WE. Evidence for the regulation of exocytic transport by protein phosphorylation. J Cell Biol. 1992;116(6):1343-55.

1315 33. Ma W, Goldberg J. TANGO1/cTAGE5 receptor as a polyvalent template for assembly of large COPII coats. Proc Natl Acad Sci U S A. 2016;113(36):10061-6.
1317 34. Townley AK, Feng Y, Schmidt K, Carter DA, Porter R, Verkade P, et al. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development. J Cell Sci. 2008;121(Pt 18):3025-34.

1320 35. Stancheva VG, Li XH, Hutchings J, Gomez-Navarro N, Santhanam B, Babu MM, et al. Combinatorial multivalent interactions drive cooperative assembly of the COPII coat. J Cell Biol. 2020;219(11).

1323 36. Hutchings J, Stancheva VG, Brown NR, Cheung ACM, Miller EA, Zanetti G. Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network. Nat Commun. 2021;12(1):2034.

1326 37. Mizoguchi T, Nakajima K, Hatsuzawa K, Nagahama M, Hauri HP, Tagaya M, et al. Determination of functional regions of p125, a novel mammalian Sec23p-interacting protein. Biochem Biophys Res Commun. 2000;279(1):144-9.

1329 38. Yang K, Liu M, Feng Z, Rojas M, Zhou L, Ke H, et al. ER exit sites in Drosophila display abundant ER-Golgi vesicles and pearled tubes but no megacarriers. Cell Rep. 2021;36(11):109707.

1332 39. Iinuma T, Aoki T, Arasaki K, Hirose H, Yamamoto A, Samata R, et al. Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains. J Cell Sci. 2009;122(Pt 10):1680-90.

1335 40. Aridor M, Fish KN, Bannykh S, Weissman J, Roberts TH, Lippincott-Schwartz J, et al. The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly. J Cell Biol. 2001;152(1):213-29.

1338 41. Bannykh SI, Rowe T, Balch WE. The organization of endoplasmic reticulum export complexes. J Cell Biol. 1996;135(1):19-35.

1340 42. Rowe T, Aridor M, McCaffery JM, Plutner H, Nuoffer C, Balch WE. COPII vesicles derived from mammalian endoplasmic reticulum microsomes recruit COPI. J Cell Biol. 1996;135(4):895-911.

1343 43. Saito K, Yamashiro K, Shimazu N, Tanabe T, Kontani K, Katada T. Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export. J Cell Biol. 2014;206(6):751-62.

1346 44. Weissman JT, Plutner H, Balch WE. The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export. Traffic. 2001;2(7):465-75.
1349 45. Itin C, Roche AC, Monsigny M, Hauri HP. ERGIC-53 is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins. Mol Biol Cell. 1996;7(3):483-93.

1352 46. Slavin I, Garcia IA, Monetta P, Martinez H, Romero N, Alvarez C. Role of Rab1b in COPII dynamics and function. Eur J Cell Biol. 2011;90(4):301-11.

1354 47. Kondylis V, Rabouille C. A novel role for dp115 in the organization of tER sites in Drosophila. J Cell Biol. 2003;162(2):185-98.

1356 48. McCaughey J, Stevenson NL, Cross S, Stephens DJ. ER-to-Golgi trafficking of procollagen in the absence of large carriers. J Cell Biol. 2019;218(3):929-48.

1358 49. Aridor M. COPII gets in shape: Lessons derived from morphological aspects of early secretion. Traffic. 2018.

1360 50. Cutrona MB, Beznoussenko GV, Fusella A, Martella O, Moral P, Mironov AA. Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport. Traffic. 2013;14(6):691-708.

1363 51. Raote I, Malhotra V. Protein transport by vesicles and tunnels. J Cell Biol. 2019;218(3):737-9.

1365 52. McCaughey J, Stevenson NL, Mantell JM, Neal CR, Paterson A, Heesom K, et al. A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function. J Cell Sci. 2021;134(17).

1368 53. Yang K, Feng Z, Pastor-Pareja JC. p24-Tango1 interactions ensure ER-Golgi interface stability and efficient transport. J Cell Biol. 2024;223(5).

1370 54. McCaughey J, Miller VJ, Stevenson NL, Brown AK, Budnik A, Heesom KJ, et al. TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion. Cell Rep. 2016;15(8):1648-59.

1373 55. Lang MR, Lapierre LA, Frotscher M, Goldenring JR, Knapik EW. Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation. Nat Genet. 2006;38(10):1198-203.

1376 56. Fromme JC, Ravazzola M, Hamamoto S, Al-Balwi M, Eyaid W, Boyadjiev SA, et al. The genetic basis of a craniofacial disease provides insight into COPII coat assembly. Dev Cell. 2007;13(5):623-34.
1379 57. Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J. Maintenance of Golgi structure and function depends on the integrity of ER export. J Cell Biol. 2001;155(4):557-70.

1382 58. Han J, Li E, Chen L, Zhang Y, Wei F, Liu J, et al. The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1. Nature. 2015;524(7564):243-6.

1384 59. Pyle E, Zanetti G. Cryo-electron tomography reveals how COPII assembles on cargo-containing membranes. bioRxiv. 2024:2024.01.17.576008.

1386 60. Shukla S, Chen W, Rao S, Yang S, Ou C, Larsen KP, et al. Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca(2+)-sensor ALG-2. Proc Natl Acad Sci U S A. 2024;121(9):e2318046121.

1389 61. Shaywitz DA, Espenshade PJ, Gimeno RE, Kaiser CA. COPII subunit interactions in the assembly of the vesicle coat. J Biol Chem. 1997;272(41):25413-6.

1391 62. Ia Cour JM, Schindler AJ, Berchtold MW, Schekman R. ALG-2 attenuates COPII budding in vitro and stabilizes the Sec23/Sec31A complex. PLoS One. 2013;8(9):e75309.

1393 63. Reuter MS, Tawamie H, Buchert R, Hosny Gebril O, Froukh T, Thiel C, et al. Diagnostic Yield and Novel Candidate Genes by Exome Sequencing in 152 Consanguineous Families With Neurodevelopmental Disorders. JAMA Psychiatry. 2017;74(3):293-9.

1397 64. Du Y, Fan X, Song C, Chang W, Xiong J, Deng L, et al. Sec23IP recruits VPS13B/COH1 to ER exit site-Golgi interface for tubular ERGIC formation. J Cell Biol. 2024;223(12).

1400 65. Pons Lanau R, Campelo F. Stairway to the Golgi: Two paths VPS13B can go by. J Cell Biol. 2024;223(12).

1402 66. Saxena S, Foresti O, Liu A, Androulaki S, Pena Rodriguez M, Raote I, et al. Endoplasmic reticulum exit sites are segregated for secretion based on cargo size. Dev Cell. 2024;59(19):2593-608 e6.

1405 67. Hanna MGt, Mela I, Wang L, Henderson RM, Chapman ER, Edwardson JM, et al. Sar1 GTPase Activity Is Regulated by Membrane Curvature. J Biol Chem. 2016;291(3):1014-27.

1408 68. Tabata KV, Sato K, Ide T, Nishizaka T, Nakano A, Noji H. Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane. EMBO J. 2009;28(21):3279-89.
1411 69. Ma W, Goldberg E, Goldberg J. ER retention is imposed by COPII protein sorting
1412 and attenuated by 4-phenylbutyrate. Elife. 2017;6.

1413 70. Copic A, Latham CF, Horlbeck MA, D'Arcangelo JG, Miller EA. ER cargo properties
1414 specify a requirement for COPII coat rigidity mediated by Sec13p. Science.
1415 2012;335(6074):1359-62.

1416 71. Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJ, Lippincott-Schwartz J.
1417 ER-to-Golgi transport visualized in living cells. Nature. 1997;389(6646):81-5.

1418 72. Venditti R, Scanu T, Santoro M, Di Tullio G, Spaar A, Gaibisso R, et al. Sedlin
1419 controls the ER export of procollagen by regulating the Sar1 cycle. Science.
1420 2012;337(6102):1668-72.

1421 73. Rowe T, Balch WE. Expression and purification of mammalian Sarl. Methods
1422 Enzymol. 1995;257:49-53.

1423