The COPII Subunit Lst1/SEC24C Acts with an Autophagy Receptor to Target

The COPII subunit Lst1/SEC24C acts with an autophagy receptor

to target endoplasmic reticulum for degradation

Yixian Cui1, Smriti Parashar1, Muhammad Zahoor2*, Patrick G. Needham3*, Muriel Mari4, Ming Zhu1, Shuliang Chen1, Hsuan-Chung Ho1, Fulvio Reggiori4, Hesso Farhan2*, Jeffrey L. Brodsky3*, and Susan Ferro-Novick1, +

1Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093-0668

2Institute of Basic Medical Sciences, Department of Molecular Medicine, University of Oslo. Sognsvannsveien 9, 0372 Oslo, Norway

3Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260

4Department of Biomedical Sciences of Cells and Systems, University of Groningen Medical Center, The Netherlands

*equal contribution

One sentence summary: Different COPII coat subunits direct ER contents onto two distinct pathways: the secretory pathway and ER-phagy

Running title: Lst1/SEC24C-mediated ER-phagy

Correspondence should be addressed to: +Dr. Susan Ferro-Novick Department of Cellular and Molecular Medicine University of California at San Diego George Palade Labs, room 315 Phone: (858) 246-0466 Fax: (858) 534-7688 e-mail: [email protected]

1 Abstract

The COPII-cargo adaptor complex, Lst1-Sec23, selectively sorts proteins into vesicles that bud from the endoplasmic reticulum (ER) and traffic to the Golgi. Improperly folded proteins are prevented from exiting the ER and are degraded. ER-phagy is an autophagic degradation pathway that utilizes ER-resident receptors. Here we report an unexpected role for Lst1-Sec23 in

ER-phagy that is independent from its function in secretion. Upregulation of the stress inducible

ER-phagy receptor, Atg40, induces the association of Lst1-Sec23 with Atg40 at distinct ER subdomains to package ER into autophagosomes. We also show that Lst1-mediated ER-phagy plays a vital role in maintaining cellular homeostasis by preventing the accumulation of aggregate-prone proteins in the ER. Lst1 function is conserved as its mammalian homologue,

SEC24C, is also required for ER-phagy.

2 Introduction

Vesicle transport cargo adaptors sort and concentrate cargo into transport vesicles at specific stages of membrane traffic (1, 2). At the ER, the Sec24 subunit of the major COPII-cargo adaptor complex, Sec24-Sec23, binds to the cytosolic domain of ER transmembrane proteins and packages them into vesicles that traffic to the Golgi (1). Yeast also has two Sec24 paralogs,

Iss1/Sfb2 (56% identity) and Lst1/Sfb3 (23% identity), that form secretory cargo adaptor complexes with Sec23, while mammals have four SEC24 isoforms. The mammalian SEC24 homologues (SEC24A and SEC24B) are 50% identical, but only share 20% identity with the

Lst1 homologues (SEC24C and SEC24D) that are also 50% identical to each other (3).

To prevent improperly folded proteins from entering the secretory pathway, the unfolded protein response (UPR) pathway works in conjunction with ER-associated degradation (ERAD) to recognize terminally misfolded proteins and retrotranslocate them into the cytosol for degradation by the proteasome (4). ERAD, however, is unable to clear all aberrant proteins from the ER (5). Macroautophagy (herein called autophagy) of the ER, or ER-phagy, could be part of a global ER stress response that restores cellular homeostasis when ERAD and/or the UPR fail to respond. There are two major types of autophagy pathways, bulk and selective (6). During starvation, bulk autophagy uses autophagosomes to scavenge cytoplasmic components for nutrients. To rapidly increase autophagosome number, COPII vesicles are rerouted to serve as a membrane source for bulk autophagy (6, 7). Selective pathways, instead, employ autophagy receptors to package cargo into autophagosomes. These cargoes include damaged or superfluous organelles, protein aggregates and pathogens (8).

Although ER-phagy was initially described in 2005 (9), it was not until the first ER- phagy receptors were identified that the process was thought to be selective (10, 11). ER-phagy

3 occurs at discrete foci on the ER (12), however, the autophagy receptors that load ER into autophagosomes are spread throughout the contiguous network of the ER. Cytosolic machinery that identifies and binds to the ER-phagy receptors may determine where autophagy sites will form. Here we report that when ER-phagy is induced, the Lst1-Sec23 complex associates with the ER-phagy receptor, Atg40, to degrade cortical ER in yeast. In the absence of Atg40 or Lst1,

ER sequestration into autophagosomes is impaired. Lst1 function in ER-phagy is conserved as its homologue, SEC24C, is required for the degradation of ER sheets and tubules in mammalian cells. Our studies have revealed a new and unexpected role for a COPII cargo adaptor complex in targeting ER domains for ER-phagy.

4 Results

Lst1 is required for ER-phagy, but not bulk autophagy

ER-phagy is typically induced in yeast by treating cells with rapamycin, a TOR kinase inhibitor that mimics nutrient starvation (9, 10). Rapamycin also upregulates the expression of Atg40, which increases ER-phagy activity (9, 10). Atg40, a reticulon-like ER membrane protein, is required for cortical ER degradation, but inefficiently degrades Hmg1 and other nuclear markers.

Nuclear ER turnover is instead predominantly facilitated by Atg39, a nucleophagy receptor (10).

As COPII coat subunits participate in membrane budding events at the ER (1), we asked if coat subunits play a role in packaging ER domains into autophagosomes during ER-phagy.

ER-phagy is induced by treating cells for 12-24 h with rapamycin, which precluded us from analyzing temperature-sensitive mutants harboring COPII mutations as they die within hours at their restrictive temperature. The lst1D, iss1D and sec24-3A mutants, however, do not significantly impair growth or secretion (13, 14). In sec24-3A cells, bulk autophagy is defective as a consequence of a failure to phosphorylate Sec24 at T324, T325, and T328 (14). Sec24 phosphorylation at these sites, which are conserved in Iss1, enables COPII coated vesicles to bind the core autophagy machinery (14). Interestingly, we found a defect in the degradation of the ER protein Sec61 in lst1D, but not in the sec24-3A iss1D and iss1D mutants (Fig. 1A-D; Fig.

S1A-C). One of the kinases that phosphorylates Sec24 during bulk autophagy, Hrr25 (14), is dispensable for ER-phagy (Fig. S1D-E). Additionally, Sec24 phosphorylation is dispensable for pexophagy (Fig. S2A-B) and mitophagy (Fig. S2D-E). The degradation of Rtn1, which marks

ER tubules, the edges of sheets and the nuclear ER in a small fraction of cells (15), was also defective in the lst1D mutant (Fig. 1E-F; Fig. S3A-B). Similarly, a third ER protein, Per33, was degraded less efficiently in lst1D cells (Fig. S3C-F). Like Sec61, Per33 resides on the nuclear

5 and cortical ER (16). In addition to rapamycin, Lst1-mediated ER degradation could also be induced by growth to the stationary phase (Fig. S4A-C).

To ask if Lst1 functions in bulk autophagy, we measured the activation of vacuolar alkaline phosphatase (Pho8D60) after starvation (Fig. S4D), as well as the cleavage of GFP-Atg8 in the vacuole (Fig. S4E-F) 24 h after rapamycin treatment (17). When bulk autophagy is induced, GFP-Atg8 and Pho8D60 are delivered to the vacuole, where Pho8D60 is activated by proteolytic cleavage. No defect in bulk autophagy was detected in lst1D cells by either assay.

Additionally, Lst1 was not needed for pexophagy (Fig. S2B-C), mitophagy (Fig. S2E-F) or the biosynthetic cytosol to vacuole targeting (Cvt) pathway (Fig. S2G), which also uses autophagy machinery (17). Therefore, Lst1 and its paralogue Sec24, appear to function in different autophagy pathways. While phosphorylation of the Sec24 membrane distal surface is required for bulk autophagy (14), it is dispensable for ER-phagy. In contrast, Lst1 acts exclusively in ER- phagy.

Lst1 is primarily known for the formation of larger COPII coated vesicles and the trafficking of GPI anchored proteins (1). Unlike Sec24, Lst1 cannot package the ER-Golgi fusion machinery (i.e., SNAREs) into COPII vesicles. Consequently, vesicles containing Lst1, but not Sec24, are unable to fuse with the Golgi (18). When we measured ER-phagy in two lst1 mutants (LST1-B1 and LST1-B2) that disrupt the packaging of GPI anchored proteins into COPII vesicles (19), no ER-phagy defect was observed (Fig. S5A-D). Thus, the role of Lst1 on the secretory pathway can be uncoupled from its contribution to ER-phagy. Together, these results establish a previously unanticipated role in autophagy for this conserved coat protein.

6 Lst1 acts in concert with Atg40

Interestingly, Sec61-GFP positive ER cisternae and foci accumulated in rapamycin-treated atg40D and lst1D mutants (Fig. S6A-B), and the ER degradation defect in the lst1Datg40D

double mutant was not significantly different than either single knockout (Fig. S6C-F). These findings imply that Lst1 and Atg40 function on the same pathway. As elevated levels of autophagy receptors increase ER-phagy and the number of ER-containing autophagosomes (10,

11), ER-phagy receptors were proposed to play a role in packaging ER into autophagosomes

(11). To address if Lst1 works in conjunction with Atg40 to perform this function, we induced

Atg40 expression with rapamycin (10) and asked if this promotes an association with Lst1. To colocalize Lst1-3xGFP with Atg40-2xmCherry, we focused on Atg40 puncta that lie within the cell interior as previous studies indicated these puncta are accessible to the autophagy machinery

(16). Sec24-GFP and Sec13-GFP, a component of the COPII coat scaffold complex (Sec13-

Sec31) (1), were examined as controls. Although all COPII coat subunits mark ER exit sites

(ERES) in nutrient-rich growth conditions (20), only Lst1-3xGFP and its partner Sec23-GFP showed increased colocalization with Atg40-2xmCherry puncta (Fig. 2A-C; Fig. S7A-C). Unlike

Atg40, Lst1 and Sec23 expression did not increase with rapamycin treatment (Fig. S7D). The number of Lst1-3xGFP and Sec23-GFP puncta were also unchanged (Fig. S7E). While previous studies showed CUP1-driven Atg40 overexpression only increased ER-phagy in the presence of rapamycin (10), overexpression was sufficient to drive Atg40 colocalization with Lst1 (Fig.

S8A-B). Rapamycin also induced the expression of the nucleophagy receptor, Atg39 (10), but did not induce the colocalization of Lst1 with Atg39 (Fig. S8C-D). Furthermore, Lst1 did not contribute to the degradation of the nuclear ER marker Hmg1 (Fig. S8E-H).

7 Selective autophagy receptors bind to the ubiquitin-like protein, Atg8, at sites of autophagosome formation (8). Consistent with the proposal that Lst1 functions with Atg40 to target ER domains for degradation, rapamycin induced Atg8-Lst1 colocalization in wild-type

(WT), but not atg40D cells (Fig. 2D; Fig. S9A). In contrast, Atg8 showed increased colocalization with two COPII coat subunits that are required for bulk autophagy, Sec24 and

Sec13 (21), in WT and atg40D cells (Fig. 2D; Fig. S9A). Because Sec23 is a binding partner for both Lst1 and Sec24, the colocalization of Atg8 with Sec23 was only partially dependent on

Atg40 (Fig. 2D; Fig. S9A).

Next, we asked whether ER organization plays a role in the colocalization of Lst1 with

Atg40. Lnp1, a transmembrane ER protein that resides at the three-way junctions, stabilizes ER network rearrangements (22). In the absence of Lnp1, Atg40 puncta fail to access the autophagy machinery (16). As a consequence, ER is not packaged into autophagosomes in lnp1D cells (16).

We found that Lst1-3xGFP failed to colocalize with Atg40-2xmCherry (Fig. 2E, Fig. S9B; also see Pearson’s coefficient in Fig. S9C) in lnp1D cells. Additionally, even though Lst1-3xGFP and

RFP-Atg8 puncta appeared unaltered in the lnp1D mutant, Lst1 also failed to colocalize with

Atg8 (Fig. 2F; Fig. S9D). We propose that Atg8-Lst1 colocalizing puncta (usually one per cell) represent active ER-phagy sites stabilized by Lnp1. Although Atg40 puncta tend to accumulate at the cell periphery and fail to reside in the cell interior in Lnp1-depleted cells (16), the distribution of Atg40 puncta is unperturbed in lst1D cells (Fig. S9E).

The localization studies described above suggested that Lst1 and Atg40 interact with each other during ER-phagy. Lst1 could interact with Atg40 via its cytosolic domain (10), however, because the Atg40 cytoplasmic domain was unstable, we could not directly test this possibility.

We were also unable to express full length Atg40 from bacteria. Therefore, to address this

8 question, bindings were performed with lysates prepared from untreated and rapamycin-treated cells (Atg40-3xFLAG). Atg40-3xFLAG from both lysates bound to purified bacterially produced

GST-Lst1, but not to GST-Sec24, GST-Sec13, or GST-Sec23 (Fig. 3A; Fig. S10A-C). GST-

Lst1 also failed to bind to two other ER proteins, Ufe1 and Yip1 (Fig. S10D). These findings indicate that Lst1, but not Sec24 or other COPII coat subunits, binds to Atg40. Sec13-Sec31 could fail to associate with Atg40 because the binding of Lst1-Sec23 to Atg40 sterically hinders its interaction. Supporting the proposal that the interaction of Atg40 with Lst1 is needed for ER- phagy, the rapamycin-induced degradation of Lst1-GFP required Atg40, and Atg40-GFP degradation required Lst1 (Fig. S11A-H).

To count ER-containing autophagosome numbers in the lst1D mutant, cells depleted of the vacuolar protease Pep4 were examined by conventional electron microscopy (EM) (Fig. 3B-

D). While autophagic bodies were absent in the atg14D mutant that blocks autophagosome formation (Fig. 3B-D), the number of autophagosomes formed in the lst1D mutant was the same as in WT (Fig. 3D). In contrast, a significant reduction in autophagic bodies that contained ER tubules and sheets was observed in the lst1D mutant (Fig. 3B-C). Consistent with the proposal that Lst1 works with Atg40 to package cortical ER into autophagosomes, the lst1D defect was comparable to that seen in atg40D cells (Fig. 3B-C).

ER stress upregulates Atg40

In mammalian cells, the putative ER-phagy receptor, FAM134B, targets ERAD-insensitive substrates to autophagy (5). How the ER communicates with the autophagy machinery when

ERAD does not respond or is overwhelmed is unknown.

9 The Z variant of human alpha-1 antitrypsin (ATZ) is an ERAD substrate that is also targeted to a non-autophagosome-mediated lysosomal pathway in mammalian cell culture (23).

When ATZ is highly overexpressed in yeast under the inducible galactose promoter (GAL1), however, the excess ATZ not degraded by ERAD is directed to the vacuole via two pathways, vacuolar protein sorting and autophagy (24). Blocking the latter pathway leads to the aggregation of ATZ in the ER (24). Currently it is unknown if this autophagosome-mediated pathway is ER- phagy. To ask whether ATZ aggregates in the absence of Atg40 and Lst1, microsomal membrane fractions prepared from WT, atg14D, atg40D and lst1D cells were analyzed on sucrose gradients.

Soluble ATZ was primarily found at the top of the WT gradient, while ATZ from mutant lysates was largely in the pellet (Fig. 4A-C; Fig. S12A-B). Aggregation appeared to be due to a block in

ER-phagy because significantly less ATZ was observed in the pellet when lysates from a mutant

(lst1-B1) that disrupts membrane traffic (19), and not ER-phagy (Fig. S5), were examined (Fig.

S12C). Aggregation was also specific to misfolded ATZ, as appreciably less wild-type M variant alpha-1 antitrypsin (ATM) was observed in pellet fractions of lst1D or atg40D cells (Fig. 4D).

Consistent with this observation, a portion of the ATM (but not ATZ) was secreted (Fig. S12D).

Together, these data indicate that only mutant alpha-1 antitrypsin aggregates in the ER in the absence of ER-phagy. Interestingly, the loss of the pancreatic ER-phagy receptor, CCPG1, also leads to the accumulation of aggregated proteins in the ER (12).

Previous studies showed that multiple COPII coat subunits, including Sec24 and Lst1, are targets of the UPR (25). To ask if Atg40 is also a UPR target, we overexpressed ATZ in the presence or the absence of Ire1, an ER transmembrane serine/threonine protein kinase that triggers the UPR (26). We found that ATZ induction enhanced Atg40 expression in WT cells

(Fig. 5A-B, compare with Fig. S12E). Additionally, Atg40 expression and ATZ accumulation

10 were further enhanced in ire1D mutant cells, which block UPR activation and augment ER stress

(4) (Fig. 5A-B). In contrast, overexpressed ATM did not increase Atg40 expression in the ire1D mutant (Fig. S12F). To determine whether the loss of Atg40 induces the UPR, we examined pathway induction by flow cytometry in cells carrying an integrated UPR-regulated GFP reporter. While the atg39D (Fig. 5C), and sec24-3A iss1D mutants behaved like WT (Fig. S12G), the level of UPR induction in the atg40D and lst1D mutants was the same as in an ERAD- deficient hrd1D strain (Fig. 5C) (4). Therefore, although Atg40 is not a UPR target, the UPR responds to the loss of ER-phagy.

As Atg40 expression is not under the control of the UPR, we next sought to address how it is regulated. Since ER stress is known to modulate the expression of core autophagy machinery

(27), we considered the possibility that autophagy transcriptional regulators control Atg40 expression. Pho23, which associates with the histone deacetylase large complex (Rpd3L), is a transcriptional repressor of autophagy that modulates autophagosome abundance via Atg9 (28).

Interestingly, Atg40, but not the UPR targets, Kar2 and Lst1, were upregulated in the rpd3D and pho23D strains (Fig. 5D-G). Atg40 and Atg9 mRNA levels also dramatically increased in these mutants (Fig. 5H), indicating that the Atg40 ER-phagy receptor is a target of the same transcriptional regulators that modulate core autophagy machinery (Atg9).

Mammalian SEC24C is required for ER-phagy

It was recently reported that SEC24A and SEC24B, but not SEC24C and SEC24D, are needed for bulk autophagy (29) To ask if any of the SEC24 isoforms are required for ER-phagy in mammalian cells, all four were knocked-down by siRNA (Fig. S13A), and the delivery of two

ER-phagy receptors, FAM134B and RTN3, to lysosomes was monitored in Torin2 (TOR

11 inhibitor2) treated cells. We chose to examine these two mammalian ER-phagy receptors because Atg40 has a domain structure that is similar to FAM134-B, yet localizes to the tubular

ER like RTN3 (11, 30). 3xFLAG-FAM134B was resistant to degradation in SEC24C-depleted cells, but not cells depleted of SEC24A, SEC24B or SEC24D (Fig. 6A; Fig. S13B). Moreover, the delivery of 3xFLAG-FAM134B to lysosomes (LAMP1) required SEC24C and ULK1, a component of the autophagosome biogenesis machinery (Fig. 6B-C; Fig. S13C). The delivery of

RTN3 to lysosomes was also SEC24C and ULK1-dependent (Fig. 6D-E; Fig. S14). Therefore, in mammals as in yeast, TOR inhibition stimulates the delivery of ER to lysosomes via

Lst1/SEC24C-mediated ER-phagy.

12 Discussion

Here we report a new role for the COPII-cargo adaptor complex, Lst1-Sec23, and show that it functions with the ER-phagy receptor, Atg40, to specify ER domains for ER-phagy. By analogy to its role as a cargo adaptor in vesicle traffic (19), Lst1-Sec23 may sort ER domains into autophagosomes via an interaction with the cytoplasmic domain of Atg40 (Fig. S15). Several lines of evidence highlight the key events mediated by Lst1 on this pathway. First, the Lst1-

Sec23 complex associates with Atg40 when its expression is upregulated by the TOR inhibitor rapamycin. Second, in accord with the proposal that the colocalization of Lst1 with Atg40 is needed for ER-phagy, Lst1 failed to colocalize with Atg40 and Atg8 in the lnp1D mutant, which disrupts ER network dynamics and the incorporation of ER into autophagosomes (16). Third, consistent with a role in ER packaging, Lst1 is required for the degradation of Atg40, and the number of ER-containing autophagosomes is significantly reduced in the lst1D mutant.

To date, six ER-phagy receptors have been identified in mammalian cells (11, 12, 30-34).

While some autophagy receptors respond to starvation, others act in response to ER stress. Here we show that Atg40 responds to both types of cellular stress. It is upregulated by both rapamycin

(10), and the overexpression of an aggregate-prone misfolded secretory protein (ATZ). In the latter case, the induction of Lst1-mediated ER-phagy reduced the level of aggregated ATZ in the

ER (Fig. S15). Consistent with the observation that a TOR inhibitor upregulates Atg40 (10), we found that Atg40 expression is modulated by autophagy transcriptional regulators. As recent studies have shown that ER stress inhibits TOR (35), TOR-dependent autophagy transcriptional regulators may also modulate the upregulation of ER-phagy receptors for the purpose of reducing ER protein aggregation.

13 Our findings imply that a specific subcomplex of the COPII coat, that contains Lst1-

Sec23, binds to Atg40 to package ER domains into autophagosomes during ER-phagy (Fig.

S15). ER-phagy occurs at distinct sites on the ER that we named ERPHS (ER-phagy sites). The absence of Sec24 from these sites distinguishes them from the ERES (ER exit sites) that bud conventional COPII vesicles (Fig. S15). Because previous studies in yeast did not reveal a significant accumulation of cytosolic ER fragments during ER-phagy, it was suggested that ER subdomains are engulfed into autophagosomes on the ER network, or in close proximity to the network (9) (Fig. S15). ER fragments appear to be packaged into autophagosomes before they completely uncoat, as we found Lst1-GFP gets degraded in the vacuole in an Atg40-dependent fashion (Fig. S15). Since the Lst1-Sec23 complex cannot package SNAREs, fragments that bud from the ER should not fuse with the Golgi prior to their incorporation into autophagosomes.

The formation of ERPHS depended on the ER protein Lnp1, which is needed for stable three-way ER junctions (Fig. S15) (22). Consistent with this observation, the mammalian ER- phagy receptor, TEX264, was recently shown to be incorporated into autophagosomes at these junctions (33, 34). The localization of LNP1 to ER junctions depends on ATLASTIN, which is also required for ER-phagy (15, 36, 37). Interestingly, ATLASTIN 3 (ATL3) was recently reported to be an ER-phagy receptor (32). Mutations in ATLASTIN and LNP1 have been linked to hereditary spastic paraplegias (HSP) and HSP-like neuropathies (38, 39). The ER-phagy receptor FAM134B, which is required for both ER-phagy and autophagosome-independent ER- to-lysosome degradation (23), has also been linked to neuronal function (11).

Additionally, we found that the mammalian Lst1 homologue, SEC24C, is required for the delivery of ER sheets (FAM134B) and tubules (RTN3) to lysosomes for degradation. This observation is in accord with recent data showing that the interactome for the ER-phagy receptor,

14 RTN3, includes SEC24C but not the other SEC24 isoforms (30). Although professional secretory cells are not sensitive to SEC24C depletion, its loss in postmitotic neurons leads to microcephaly and perinatal death (40). While SEC24C and SEC24D are ~50% identical, their functions only partially overlap in the brain (40), and we did not detect a significant role for SEC24D in ER- phagy. As our experiments and the RTN3 interactome studies both used U2OS cells, it remains formally possible that other cell types may require SEC24D for ER-phagy. Together, these findings suggest that ER-phagy plays a crucial role in maintaining neuronal homeostasis.

In summary, we propose that in yeast and mammals, COPII cargo adaptors package different cargoes (secretory proteins or ER domains) into membranes and target theses cargoes to distinct pathways. Our findings imply that ERES, which bud canonical COPII vesicles on the secretory pathway, are distinct from the ERPHS used in autophagy. The secretory and ER-phagy pathways also appear to be regulated by different stress response pathways.

15 Figure Legends

Figure 1. Lst1 is required for ER-phagy.

(A) The translocation of Sec61-GFP to the vacuole was examined by fluorescence microscopy in

WT and mutant cells 24 h after rapamycin treatment. FM4-64 was used to stain the vacuolar membrane. (B) The percent of cells with Sec61-GFP delivered to the vacuole was quantitated from 300 cells. (C) The cleavage of Sec61-GFP to GFP in WT and mutant strains was analyzed by immunoblotting using anti-GFP antibody. A representative blot is shown. (D) Three separate experiments were used to quantitate the ratio of free GFP to Sec61-GFP from the data in (C).

WT was set to 100%. (E) The translocation of Rtn1-GFP to the vacuole in WT and mutant cells was detected by fluorescence microscopy 24h after rapamycin treatment. (F) The percent of cells with Rtn1-GFP delivered to the vacuole was quantitated from 300 cells. Scale bars in (A), (E),

5µm. Error bars in (B), (D), and (F) represent S.E.M., N=3; *P < 0.05, **P < 0.01, ***P < 0.001,

Student’s unpaired t-test.

Figure 2. Lst1 and Sec23 colocalize with Atg40 and Atg8 in rapamycin-treated cells.

(A) Representative images of cells treated for 0 or 6 h with rapamycin. Arrowheads indicate

Lst1-3xGFP that colocalize with Atg40-2xmCherry puncta. (B) Bar graph shows the percent of

Lst1-3xGFP or Sec24-GFP colocalizing with Atg40-2xmCherry puncta. (C) Bar graph shows the percent of Sec23-GFP or Sec13-GFP colocalizing with Atg40-2xmCherry puncta. (D) Cells were treated for 0 or 3 h with rapamycin, and RFP-Atg8 that colocalizes with GFP tagged COPII coat subunits was quantitated. (E) Left, arrowheads indicate Lst1-3xGFP that colocalizes with Atg40-

2xmCherry puncta 6 h after rapamycin treatment. Quantitation is on the right. (F) Quantitation of

RFP-Atg8 that colocalize with Lst1-3xGFP in WT and lnp1D cells. Scale bars in (A), (E), 5µm.

16 Error bars in (B-F) represent S.E.M., N=3-6; NS, not significant P > 0.05, *P < 0.05, **P < 0.01,

***P < 0.001, Student’s unpaired t-test.

Figure 3. Lst1 binds to Atg40 and facilitates the packaging of ER into autophagosomes.

(A) Equimolar amounts (0.1 µM) of purified recombinant GST and GST fusion proteins were incubated with 2mg of yeast lysate prepared from Atg40-3xFLAG untagged or tagged cells treated with rapamycin. (B) Representative electron micrographs of WT and pep4D strains treated for 12 h with rapamycin. The boxed areas in the left panel are magnified on the right.

Black asterisk, an autophagic body containing an ER fragment; white asterisk, an autophagic body lacking an ER fragment; arrowhead, an ER fragment inside an autophagic body. Scale bars in the left and right panels represent 1µm and 500 nm, respectively. (C) Bar graph showing the average number of ER-containing autophagosomes. (D) Bar graph showing the average number of total autophagosomes. Error bars in (C) and (D) represent S.E.M., N=100 cells; *P < 0.05,

***P < 0.001, Student’s unpaired t-test. Symbols: N, nucleus; V, vacuole.

Figure 4. ATZ aggregates accumulate in atg40D and lst1D mutants.

Microsomal membrane fractions prepared from WT and mutant cells, harboring plasmids expressing either ATZ (A-D) or ATM (D), were lysed in the presence of detergent and fractionated on a sucrose gradient. Gradient fractions were blotted with antibody directed against alpha-1 antitrypsin. Soluble alpha-1 antitrypsin was at the top of the gradient and aggregates reside in the pellet (P).

17 Figure 5. Atg40 expression is regulated by the Rpd3-Pho23 complex.

(A) Atg40 expression was examined 24 h after growth in 2% galactose. Western blot analysis was performed on lysates to detect ATZ (top), Atg40-3xFLAG (middle) and Adh1 (bottom). (B)

The data in (A) were normalized to Adh1, and WT (-ATZ) was set to 100%. (C) UPR induction was assayed by flow cytometry in the absence or presence of 8mM DTT. WT (-DTT) was set to

1.0. (D) Atg40, Kar2 and Lst1 levels were measured by western blot analysis in the indicated strains. (E-G) The data in (D) were normalized to Adh1 and WT was set to 100%. (H) qRT-PCR was used to assess mRNA levels. The data were normalized to actin mRNA levels, and WT was set to 1.0. Error bars in (B, C, and H) represent S.E.M., N=3; NS, not significant, P > 0.05. Error bars in (E) represent S.E.M., N=8. Error bars in (F, G) represent S.E.M., N=4. *P < 0.05, **P <

0.01, ***P < 0.001, Student’s unpaired t-test.

Figure 6. SEC24C is required for ER-phagy.

(A) Quantitation of 3xFLAG-FAM134B degradation from Torin2 treated control siRNA or

SEC24 siRNA depleted U2OS cells normalized to actin. (B and D) Representative images of siRNA depleted cells treated with Torin2 and Bafilomycin A1. Quantitation of 3x-FLAG-

FAM134B (C) or RTN3 (E) in LAMP1 structures. The DMSO control for each condition was set to 1.0. The data for two different siSEC24C duplexes is shown in C. Scale bar in (B and D) is 10µm. Error bars in (A, C, E) represent S.E.M., N=3-5, 50-70 cells/experiment for FAM134B and 30 cells/experiment for RTN3; NS, not significant P > 0.05, *P < 0.05, **P < 0.01, ***P <

0.001, Student’s unpaired t-test.

References

1. N. Gomez-Navarro, E. Miller, Protein sorting at the ER-Golgi interface. J Cell Biol 215, 769-778 (2016). 2. T. Kirchhausen, J. S. Bonifacino, H. Riezman, Linking cargo to vesicle formation: receptor tail interactions with coat proteins. Curr Opin Cell Biol 9, 488-495 (1997). 3. B. L. Tang, J. Kausalya, D. Y. Low, M. L. Lock, W. Hong, A family of mammalian proteins homologous to yeast Sec24p. Biochem Biophys Res Commun 258, 679-684 (1999). 4. P. Walter, D. Ron, The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081-1086 (2011). 5. A. Forrester et al., A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex. EMBO J 38, (2019). 6. S. Davis, J. Wang, S. Ferro-Novick, Crosstalk between the Secretory and Autophagy Pathways Regulates Autophagosome Formation. Dev Cell 41, 23-32 (2017). 7. T. Shima, H. Kirisako, H. Nakatogawa, COPII vesicles contribute to autophagosomal membranes. J Cell Biol, (2019). 8. A. L. Anding, E. H. Baehrecke, Cleaning House: Selective Autophagy of Organelles. Dev Cell 41, 10-22 (2017). 9. M. Hamasaki, T. Noda, M. Baba, Y. Ohsumi, Starvation triggers the delivery of the endoplasmic reticulum to the vacuole via autophagy in yeast. Traffic 6, 56-65 (2005). 10. K. Mochida et al., Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus. Nature 522, 359-362 (2015). 11. A. Khaminets et al., Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522, 354-358 (2015). 12. M. D. Smith et al., CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis. Dev Cell 44, 217-232 e211 (2018). 13. K. J. Roberg, M. Crotwell, P. Espenshade, R. Gimeno, C. A. Kaiser, LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum. J Cell Biol 145, 659-672 (1999). 14. S. Davis et al., Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation. eLife 5, (2016). 15. S. Chen, P. Novick, S. Ferro-Novick, ER network formation requires a balance of the dynamin-like GTPase Sey1p and the Lunapark family member Lnp1p. Nature cell biology 14, 707-716 (2012). 16. S. Chen, Y. Cui, S. Parashar, P. J. Novick, S. Ferro-Novick, ER-phagy requires Lnp1, a protein that stabilizes rearrangements of the ER network. Proc Natl Acad Sci U S A, (2018). 17. D. J. Klionsky, A. M. Cuervo, P. O. Seglen, Methods for monitoring autophagy from yeast to human. Autophagy 3, 181-206 (2007). 18. E. Miller, B. Antonny, S. Hamamoto, R. Schekman, Cargo selection into COPII vesicles is driven by the Sec24p subunit. EMBO J 21, 6105-6113 (2002). 19. E. A. Miller et al., Multiple cargo binding sites on the COPII subunit Sec24p ensure capture of diverse membrane proteins into transport vesicles. Cell 114, 497-509 (2003).

19 20. H. Iwasaki, T. Yorimitsu, K. Sato, Distribution of Sec24 isoforms to each ER exit site is dynamically regulated in Saccharomyces cerevisiae. FEBS Lett 589, 1234-1239 (2015). 21. M. Hamasaki, T. Noda, Y. Ohsumi, The early secretory pathway contributes to autophagy in yeast. Cell Struct Funct 28, 49-54 (2003). 22. S. Chen et al., Lunapark stabilizes nascent three-way junctions in the endoplasmic reticulum. Proc Natl Acad Sci U S A 112, 418-423 (2015). 23. I. Fregno et al., ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport. EMBO J 37, (2018). 24. K. B. Kruse, J. L. Brodsky, A. A. McCracken, Characterization of an ERAD gene as VPS30/ATG6 reveals two alternative and functionally distinct protein quality control pathways: one for soluble Z variant of human alpha-1 proteinase inhibitor (A1PiZ) and another for aggregates of A1PiZ. Molecular biology of the cell 17, 203-212 (2006). 25. K. J. Travers et al., Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101, 249-258 (2000). 26. J. S. Cox, C. E. Shamu, P. Walter, Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73, 1197-1206 (1993). 27. T. Yorimitsu, U. Nair, Z. Yang, D. J. Klionsky, Endoplasmic reticulum stress triggers autophagy. J Biol Chem 281, 30299-30304 (2006). 28. M. Jin et al., Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation. Curr Biol 24, 1314-1322 (2014). 29. Y. T. Jeong et al., The ULK1-FBXW5-SEC23B nexus controls autophagy. eLife 7, (2018). 30. P. Grumati et al., Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. eLife 6, (2017). 31. F. Fumagalli et al., Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery. Nature cell biology 18, 1173-1184 (2016). 32. Q. Chen et al., ATL3 Is a Tubular ER-Phagy Receptor for GABARAP-Mediated Selective Autophagy. Curr Biol 29, 846-855 e846 (2019). 33. H. Chino, T. Hatta, T. Natsume, N. Mizushima, Intrinsically Disordered Protein TEX264 Mediates ER-phagy. Mol Cell, (2019). 34. H. An et al., TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress. Mol Cell, (2019). 35. L. Luo et al., De-silencing Grb10 contributes to acute ER stress-induced steatosis in mouse liver. J Mol Endocrinol 60, 285-297 (2018). 36. J. R. Liang, E. Lingeman, S. Ahmed, J. E. Corn, Atlastins remodel the endoplasmic reticulum for selective autophagy. J Cell Biol 217, 3354-3367 (2018). 37. S. Wang, H. Tukachinsky, F. B. Romano, T. A. Rapoport, Cooperation of the ER-shaping proteins atlastin, lunapark, and reticulons to generate a tubular membrane network. eLife 5, (2016). 38. J. K. Fink, Hereditary spastic paraplegia: clinico-pathologic features and emerging molecular mechanisms. Acta Neuropathol 126, 307-328 (2013). 39. M. W. Breuss et al., Mutations in LNPK, Encoding the Endoplasmic Reticulum Junction Stabilizer Lunapark, Cause a Recessive Neurodevelopmental Syndrome. Am J Hum Genet 103, 296-304 (2018).

20 40. B. Wang et al., The COPII cargo adapter SEC24C is essential for neuronal homeostasis. J Clin Invest 128, 3319-3332 (2018).

Acknowledgments

We thank Shuaipeng Ma for technical assistance during the early phase of these studies, Randy Hampton for the 4xUPRE-GFP plasmid and the use of his flow cytometer, Elizabeth Miller for plasmids, and Andrea Lougheed for assistance in the preparation of figures. Funding: Salary support for S.F-N., Y.C., S.P., M.Z., S.C. and H.-C.H. was provided by the ALPHA-1 FOUNDATION and NIGMS under award numbers R01GM114111, R01GM115422 and R35GM131681. Salary support for P.G.N. and J.L.B was provided by grants P30DK79307 and 1R01GM75061. H.F. is supported by the Norwegian Research Council, the Norwegian Cancer Society, and the Ander Jahre Foundation. F.R. is supported by Marie Skłodowska-Curie Cofund (713660), Marie Skłodowska-Curie ITN (765912), ALW Open Program (ALWOP.310), ZonMW VICI (016.130.606) and ZonMW TOP (91217002) grants. M.M. is supported by an ALW Open Program grant (ALWOP.355). Author contributions: Y.C., S.P., M.Z., P.G.N., M.M., M.Z., S.C., H.-C.H., F.R., H.F., J.L.B. and S.F.-N. designed research and analyzed data; Y.C., S.P., M.Z., P.G.N., M.M., M.Z., S.C. and H.-C.H., performed research; and S.F.-N., J.L.B., F.R., Y.C., S.P., M.M. and H.F. wrote the paper. Competing interests: Authors declare no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials.

Supplementary Materials: Materials and methods Figures S1-S15 Tables S1-S2 References

21 A B Sec61-GFP FM4-64 Merge

70 atg40Δ 60

40 * lst1Δ * 30 /Total cells (%) /Total 20

10 sec24-3A iss1Δ vacuole in Sec61-GFP with Cells 0 Δ Δ Δ Δ WT lst1 iss1 atg40

iss1Δ sec24-3A iss1

C D 120 WT atg40Δ lst1Δ Rapamycin (h): 024 024 0 24 100 age v Sec61-GFP 80

60 WTclea **

40 ** GFP 20 Adh1 Percent of 0 Δ Δ WT lst1 atg40

Rtn1-GFP FM4-64 Merge 80 WT 70 60 50 40 atg40Δ 30 ***

/Total cells (%) /Total 20 *** 10

lst1Δ vacuole in Rtn1-GFP with Cells 0 Δ Δ WT lst1 atg40 Figure 1 A B C Atg40- Lst1-3xGFP Sec23-GFP Lst1-3xGFP 2xmCherry Merge 30 Sec24-GFP 30 Sec13-GFP ** 0 h 25 25 20 *** 20 ***

15 15 ***

10 10 6 h

puncta colocalized with 5 puncta colocalized with 5 GFP GFP Atg40-2xmCherry puncta (%) 0 Atg40-2xmCherry puncta (%) 0 036 036 Rapamycin (h) Rapamycin (h)

Lst1 Sec24 Sec23 Sec13 NS 30 30 30 30 ** ** ** 25 25 *** 25 25 * NS *** *** *** WT 20 WT 20 20 20 ** NS atg40Δ atg40Δ 15 15 15 15 WT WT 10 10 10 atg40Δ 10 atg40Δ 5 5 5 5 with Sec24-GFP puncta (%) with Sec24-GFP puncta (%) with Sec23-GFP puncta (%) with Sec13-GFP with Lst1-3xGFP puncta (%) with Lst1-3xGFP RFP-Atg8 puncta colocalized 0 RFP-Atg8 puncta colocalized 0 RFP-Atg8 puncta colocalized 0 RFP-Atg8 puncta colocalized 0 03 03 03 03 Rapamycin (h) Rapamycin (h) Rapamycin (h) Rapamycin (h)

E F Atg40- Lst1-3xGFP 2xmCherry Merge 30 WT 30 WT lnp1Δ lnp1Δ 25 *** 25 WT ** 20 *** 20 15 15

10 10 lnp1 5 5 with Lst1-3xGFP puncta (%) with Lst1-3xGFP RFP-Atg8 puncta colocalized

Lst1-3xGFP puncta colocalized Lst1-3xGFP 0 0

with Atg40-2xmCherry puncta (%) 03 6 03 Rapamycin (h) Rapamycin (h)

Figure 2 A Input

-Lst1 -Lst1 -Sec13-Sec13-Sec24-Sec24 T T T T T T 0.1% 0.1% 0.05%0.025%GST GST GS GS GS GS GS GS Untagged lysate + - - - + - + - + - + - Atg40-3xFLAG lysate - + + + - + - + - + - +

Atg40-3xFLAG

Lane 1 2 3 4 5 6 7 8 9 10 11 12

N ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ V V V N WT ∗ atg40∆ ∗ ∗ ∗ V ∗ ∗ ∗ ∗ ∗ ∗

V V ∗∗ ∗ atg14∆ lst1∆ ∗ ∗ ∗ ∗ N V ∗ V ∗ N ∗

C D 1.4 9 8 1.2 7 1 * 6 0.8 *** *** 5 0.6 4 3

0.4 bodies per section 2 0.2 autophagic bodies per section

Average number of autophagic Average 1 Average number of ER-containing Average 0 *** 0 *** WT WT lst1∆ lst1∆ atg14∆ atg40∆ atg14∆ atg40∆

Figure 3 A

35 WT atg14 30

ATZ (%) ATZ 15

0 12345678910111213P Fraction Number

B C

50 WT Fraction number 45 atg40 1 2 3 4 5 6 7 8 9 10 11 12 13 P 40 lst1 ATZ WT 35 30 1 2 3 4 5 6 7 8 9 10 11 12 13 P 25 atg40 ATZ (%) ATZ 20

1 2 3 4 5 6 7 8 9 10 11 12 13 P 15 10 lst1 ATZ 5 0 12345678910111213P Fraction Number

D 35 WT ATM 35 WT ATM WT ATZ WT ATZ 30 30 atg40 ATM lst1 ATM 25 atg40ATZ 25 lst1 ATZ

20 20

15 15 ATM or ATZ (%) ATZ or ATM (%) ATZ or ATM 10 10

5 5

0 0 12345678910111213P 12345678910111213P Fraction Number Fraction Number

Figure 4 A B +Galactose 350 Δ Δ ** WT ire1 WT ire1 300 Galactose ++++ ATZ-pYES2 --++ 250

ATZ 200 ** 150 * Atg40-3xFLAG 100 Atg40-3xFLAG Level (%) Level Atg40-3xFLAG Adh1 50

Lane 1432 0 ATZ-pYES2 --++ Δ Δ WT WT C ire1 ire1 30 on

ti 25 * duc * n 20 * r i te r

o 15 ** 10 ***

UPR rep UPR *

d * l 5

o **

F NS 0 DTT -----+++++ WT hrd1Δ atg39Δ atg40Δ lst1Δ

D E F G

Δ 300 300 300 Δ WT rpd3 pho23 250 250 250 *** *** Atg40-3xFLAG 200 200 200

Kar2 150 150 150 Lst1 level (%) 100 Kar2 level (%) 100 100 Lst1

Atg40-3xFLAG level (%) 50 50 50 Adh1 0 0 0 Δ Δ Δ Δ Δ Δ Lane 321 WT WT WT rpd3 rpd3 rpd3 pho23 pho23 pho23

H 14 WT 12 ** rpd3Δ pho23Δ 10 * 8

6 ** mRNA level mRNA 4 ** 2

0 ATG40 ATG9 Figure 5 A 1.2

1 **

0.8

0.6

0.4

Fraction of FAM134B 0.2

0 siSEC24 --ABCD Torin2 - +++++

B C

FAM134B LAMP1 Merge Inset siCtrl+Torin2 3 FAM134B * 2.5 *** * siSEC24C +Torin2 2

1.5 NS NS NS 1

Relative pixel intensity 0.5 siSEC24B +Torin2 0

siCtrl ULKi

siSEC24B siSEC24D siCtrl+Torin2 ULKi+Torin2siSEC24C#1 siSEC24C#2

siSEC24B+Torin2siSEC24D+Torin2 siSEC24C#1+Torin2siSEC24C#2+Torin2

DE RTN3 LAMP1 Merge Inset 3 RTN3 2.5 NS *** * ** 2 siCtrl+Torin2 1.5 NS

Relative pixel intensity 0.5 siSEC24C +Torin2 0

siCtrl

siSEC24B siSEC24D siCtrl+Torin2ULKi+Torin2siSEC24C#1

siSEC24B+Torin2siSEC24D+Torin2 siSEC24C#1+Torin2 Figure 6 A 100 WT 90 sec24-3A iss1Δ 80 70

60 50 40 /Total cells (%) cells /Total 30

20 Cells with Sec61-GFP in vacuole Cells with Sec61-GFP 10

0 13 16 20 24 Rapamycin (h)

B C

140 sec24-3A WT atg40Δ iss1Δ iss1Δ 120 Rapamycin (h): 024 024 0 24 0 24 100 Sec61-GFP 80

GFP 40 ** Percent of WTcleavage

20 Adh1

0 Δ Δ Δ WT iss1 atg40

sec24-3A iss1

DE 140

WT atg40Δ hrr25-5 120 Rapamycin (h): 024 024 0 24 Sec61-GFP 100

GFP 40 Percent of WTcleavage

20 Adh1 *** 0 Δ WT atg40 hrr25-5 Figure S1 AB Pex14-GFP DIC Merge

WT 140 100 90 sec24-3A 120 80 WT iss1Δ atg36Δ 100 70 cleavage atg36Δ SD-N (h): 000666 T 80 60 Pex14-GFP 60 50 40

nt of W 40 30 e sec24-3A **

rc 20 20 GFP e iss1Δ Cells with Pex14-GFP Pex14-GFP with Cells P 0 *** 10 Δ Δ (%) cells vacuole/Total in 0 WT Δ Δ Δ atg36 lst1Δ WT lst1 atg36

sec24-3A iss1 sec24-3A iss1

C 120 100 WT atg36Δ lst1Δ 80 SD-N (h): 066 06 0 Pex14-GFP 60 40 20 GFP Percent of WTcleavage 0 *** Δ Δ WT lst1 atg36

DE Om45-GFP DIC Merge

WT sec24-3A 140 100 WT iss1Δ atg32Δ 90 120 YPL (h): 007272 0 72 80 100 70 Om45-GFP atg32Δ 80 60 50 60 40 40 * sec24-3A 30 20 iss1Δ 20 Cells with Om45-GFP Om45-GFP with Cells GFP Percent of WT cleavage 0 10

in vacuole/Total cells (%) in vacuole/Total *** Δ Δ 0 WT Δ Δ Δ atg32 WT lst1 lst1Δ atg32

sec24-3A iss1 sec24-3A iss1

FG 200

WT atg32Δ lst1Δ age 180 160 YPL (h): 072 072 0 72 Δ eav 140 Δ cl

Om45-GFP WT atg1 lst1

T 120 100 W prApe1 80 of t 60 mApe1

en 40 c 20 ***

GFP Per 0 Δ Δ WT lst1 atg32 Figure S2 AB

120 WT atg40Δ lst1Δ 100 Rapamycin (h): 0002424 24 80 Rtn1-GFP 60 * 40 GFP * 20 Percent of WT cleavage WT of Percent Adh1 0 Δ Δ WT lst1 atg40

Per33-GFP FM4-64 Merge

80 WT 70 60 50 40 atg40Δ ** ** 30

/Total cells (%) /Total 20 10

lst1Δ vacuole in Per33-GFP with Cells 0 Δ Δ WT lst1 atg40

E F

WT atg40Δ lst1Δ 120 Rapamycin (h): 012 012 0 12 100 age

Per33-GFP v 80

60 ** 40 * GFP 20 Percent of WTclea

Adh1 0 Δ Δ WT lst1 atg40

Figure S3 AB C

120

Δ Δ 100 WT lst1 WT lst1 OD : 0.5 8.00.58.0 Sec61-GFP FM4-64 Merge 600 80 Sec61-GFP 60 WT

GFP cleavage WT of Percent 20 ** lst1Δ Adh1 0 WT lst1Δ

D 120

100

Relative Pho8 activity (%) 20 **

0 Δ Δ WT atg1 lst1

EF 120

100 WT atg1Δ lst1Δ Rapamycin (h): 00024 2424 80 GFP-Atg8 60

40 GFP

Adh1 -Atg8) (%) GFP/(GFP+GFP 20 *** 0 Δ Δ WT atg1 lst1

Figure S4 A B Sec61-GFP FM4-64 Merge

WT 100 90 80 70 lst1Δ 60 50 40 ***

lst1Δ+LST1 (%) cells /Total 30 20 10 Cells with Sec61-GFP in vacuole Cells with Sec61-GFP lst1Δ+LST1-B1 0 Δ WT B1 B2 lst1 +LST1 Δ +LST1- +LST1- lst1 Δ Δ lst1Δ+LST1-B2 lst1 lst1

C lst1Δ lst1Δ lst1Δ WT atg40Δ lst1Δ +LST1 +LST1-B1 +LST1-B2

Rapamycin (h): 0002424 24 0002424 24

Sec61-GFP Sec61-GFP

GFP GFP

Adh1 Adh1

120

100 age 80 cleav T 60 *

W * of t 40 en c

er 20 P 0 Δ Δ WT B1 B2 lst1 +LST1 atg40 Δ +LST1- +LST1- lst1 Δ Δ lst1 lst1 Figure S5 A B 80 0 h Rapamycin (h) 70 12 h *

012 0 12 60

50 * WT atg40Δ ** ** * 40 * * 30 NS * * * * 20 atg14Δ lst1Δ * * * cytoplasmic accumulating Cells (%) tubules and foci 10 * * 0 WT atg14Δ atg40Δ lst1Δ

Sec61-GFP FM4-64 Merge

90 WT 80 70 NS 60 atg40Δ NS 50 40

/Total cells (%) cells /Total 30

lst1Δ 20 10 Cells with Sec61-GFP in vacuole 0 Δ Δ Δ WT lst1 atg40 atg40 lst1Δatg40Δ Δ lst1

E F 120 lst1Δ WT atg40Δ lst1Δ atg40Δ 100 Rapamycin (h): 024 024 0 24 0 24 80 NS Sec61-GFP NS 60

40 GFP Percent of WT cleavage WT of Percent 20 Adh1

0 Δ Δ Δ WT lst1 atg40 atg40 Δ lst1

Figure S6 A B

Atg40- Atg40- Sec23-GFP2xmCherry Merge Sec24-GFP 2xmCherry Merge

0 h 0 h

6 h 6 h

C D Atg40- Sec13-GFP2xmCherry Merge 250 0 h Rapamycin (h): 0 3 3 h 200 * 0 h Lst1 150 Sec23 100 Atg40-3xFLAG Protein level (%) 50 6 h Adh1 0 Atg40 Lst1 Sec23

10 Lst1 9 Sec23 Sec24 8 Sec13 7 6 5 4 3 2 Number of GFP puncta/cell Number of GFP 1 0 0 3 6 Rapamycin (h)

Figure S7 ABLst1- Atg40-GFP 2xmCherry Merge 30

25 endo 0 h 20

15 *** *** OE 0 h 10

5 Atg40-GFP puncta colocalized with Lst1-2xmCherry puncta (%)

OE 3 h 0 CUP1-Atg40-GFP - ++ Copper (h) 0 0 3

CDAtg39- Lst1-3xGFP 2xmCherry Merge 5 4.5

0 h 4 3.5 3 2.5 3 h 2 1.5 1

Lst1-3xGFP puncta colocalized Lst1-3xGFP 0.5 with Atg39-2xmCherry puncta (%) with 6 h 0 036 Rapamycin (h)

180 E WT atg39Δ lst1Δ F

e 160 Rapamycin (h): 024 024 0 24

vag 140 Hmg1-GFP NS 120 clea

100 WT

f 80 o

nt 60

GFP erce 40 P 20 Adh1 *** 0 Δ Δ WT lst1 GHHmg1-GFP FM4-64 Merge atg39 70

60 NS WT cuole a

) 50 (% P in P v

F 40 -G

g1 30 atg39Δ Total cells cells Total / Hm

h 20

wit 10 ls

Cel *** 0 lst1Δ Δ Δ WT lst1 atg39 Figure S8 A RFP-Atg8Lst1-3xGFP Merge RFP-Atg8 Sec24-GFP Merge

WT WT

atg40Δ atg40Δ

RFP-Atg8 Sec23-GFP Merge RFP-Atg8Sec13-GFP Merge

WT WT

atg40Δ atg40Δ

B C

Sec24/Atg40 0.35 0.35 30 WT 0.3 0.3 25 lnp1 WT 0.25 0.25 20 0.2 0.2 15 0.15 * 0.15 10 0.1 0.1 lnp1 5 0.05 0.05 Coefficient of Sec24-GFP Coefficient Coefficient of Lst1-3xGFP Coefficient 0 0

Sec24-GFP puncta colocalized Sec24-GFP 0 colocalized with Atg40-2xmCherry colocalized with colocalized with Atg40-2xmCherry with colocalized with Atg40-2xmCherry puncta (%) 03 6 WT lnp1 WT lnp1 Rapamycin (h)

D E RFP-Atg8Lst1-3xGFP Merge Atg40-3xGFP Atg40-GFP WT lnp1Δ WT lst1Δ

lnp1Δ 3.0 3.0 NS 2.5 2.5

2.0 2.0

1.5 1.5

Puncta/cell 1.0 Puncta/cell 1.0 ** 0.5 0.5

0.0 0.0 WT lnp1Δ WT lst1Δ

Figure S9 A B

Input

T-Lst1 GST GST GST-Lst1GS GST-Sec13GST-Sec13GST-Sec24GST-Sec24 0.5% 0.25% GST-Sec23GST-Lst1 Untagged lysate + - + - + - + - Atg40-3xFLAG lysate ++ ++ Atg40-3xFLAG lysate +- +-+- - + Atg40-3xFLAG * * * *

Ponceau Ponceau

Lane 87654321

Lane 1 2 43

C -Lst1 -Lst1

GST GST GST GST Atg40-3xFLAG lysate + + + + Rapamycin - + - + lnput -Lst1 T * 0.05% 0.1% GST GST GST-Lst1 GS Atg40-3xFLAG lysate + + + + ++++ Rapamycin - + - + +- - + Ponceau

Atg40-3xFLAG

Lane 12 34 765 8

Lane 12 34

-Lst1 D T

GST GST GS GST-Lst1 Atg40-3xFLAG lysate + + + + Rapamycin - + - + lnput 0.05% 0.1% * GST GST GST-Lst1 GST-Lst1 Atg40-3xFLAG lysate + + + + ++ ++ Rapamycin - + - + +- - + Ponceau Ufe1

Yip1

Lane 1234 5 76 8 Lane 12 34

Figure S10 A B Lst1-3xGFP FM4-64 Merge 100 90 WT 80 70 60 50 atg1Δ 40 30 ** 20 Cells with Lst1-3xGFP with Cells in vacuole/Total cells (%) in vacuole/Total 10 *** atg40Δ 0 Δ Δ WT atg1 atg40

CDWT atg1Δ atg40Δ 120 Rapamycin (h): 024 024 0 24 100 Lst1-GFP 80

20 ** GFP cleavage WT of Percent *** 0 Adh1 Δ Δ WT atg1 atg40

EF Atg40-GFP FM4-64 Merge 100 90 WT 80 70 60 50 atg14Δ 40 ** 30 20 Cells with Atg40-GFP Atg40-GFP Cells with in vacuole/Total cells (%) cells vacuole/Total in 10 *** lst1Δ 0 Δ Δ WT lst1 atg14

GH 120 WT atg14Δ lst1Δ 100 Rapamycin (h): 0002424 24 Atg40-GFP 80 60

40 GFP ** 20 Adh1 cleavage WT of Percent 0 *** Δ Δ WT lst1 atg14

Figure S11 A B Fraction number

atg14 atg14 1 2 3 4 5 6 7 8 9 10 11 12 13 P ATZ-pYES2 ++ ATZ WT Galactose + - ATZ 1 2 3 4 5 6 7 8 9 10 11 12 13 P Adh1 atg14 ATZ Lane 12

C D 35 WT WT atg40 lst1 30 ATM ATZ ATM ATZ lst1-B1 25 TTEETTEE

20 ATM or ATZ

ATZ (%) ATZ 15

5 Pdi1 0 Lane 321 4 765 8 12345678910111213P Fraction Number

E -Galactose 350

300

250 WT 1 WT 1 Galactose ---- 200

ATZ-pYES2 --++ 150

Atg40-3xFLAG 100

Atg40-3xFLAG Level (%) 50 Adh1 0 Lane 1 32 4 ATZ-pYES2 --++

WT 1 WT 1

F G +Galactose 1 350 10 Galactose ++ 300 9 ATM-pYES2 8 -+ 250 7 ATM 200 6 5 NS 150 Atg40-3xFLAG 4 * * 100 3 2

Adh1 Atg40-3xFLAG Level (%) 50 1 Fold UPR reporter induction 0 0 _ _ Lane 21 DTT + + ATM-pYES2 - + SEC24 sec24-3A iss1 1

Figure S12 A

1.8 1.6 1.4 1.2 1.0 0.8

mRNA level mRNA 0.6 0.4 0.2 0.0 Sec24A Sec24B Sec24C Sec24D Sec24A Sec24B Sec24C Sec24D Sec24A Sec24B Sec24C Sec24D Sec24A Sec24B Sec24C Sec24D Sec24A Sec24B Sec24C Sec24D siRNA Ctrl Sec24A Sec24B Sec24C Sec24D

siSec24 --ABCD Torin2 - +++++ 3xFLAG-FAM134B

Actin

C FAM134B LAMP1 Merge Inset siCtrl+DMSO siCtrl+Torin2+ULKi Torin2 siSec24D+ Figure S13 RTN3 LAMP1 Merge Inset siCtrl+DMSO siCtrl+Torin2+ULKi siSEC24B+Torin2 siSEC24D+Torin2

Figure S14