A Role for Macro-ER-Phagy in ER Quality Control

Summary

ER-quality control (ERQC) ensures delivery of “native” proteins through the secretory pathway. Currently, ER-associated degradation (ERAD), which delivers misfolded proteins for degradation by the proteasome, is considered a major ERQC pathway, with autophagy as its backup. Until now, the role of autophagy, which shuttles cellular components for degradation in the lysosome through autophagosomes (APs), in ERQC was ill defined. Recently, the process of ER degradation induced by ER stress was defined as micro-ER-phagy, which does not require autophagic machinery and does not pass through APs. Here, we characterize the macro-ER-phagy pathway, which delivers excess membrane proteins for degradation in the lysosome, as a novel ERQC pathway. This pathway functions in the absence of cellular or ER stress and can be further induced by overexpression of a single integral-membrane protein. Unlike the micro-ER-phagy pathway, the marco-ER-phagy pathway requires core autophagy-specific proteins, Atgs, and Ypt/Rab GTPases. In addition, for the first time, the function of the only membrane core Atg, Atg9, was uncoupled from that of the other core Atgs. Whereas Atg9 plays a role in the assembly of ER-to-autophagy membranes (ERAM), other core Atgs and Ypt1 assemble the Atg-protein complex on ERAM to form the pre-autophagosomal structure.

Introduction

One third of all newly synthesized proteins enter the endoplasmic reticulum (ER). However, only a small fraction is transported to their final destination. A large fraction (30–75%) fails to fold and mature properly, does not pass the ER quality control (ERQC) and gets degraded [1]. Two different cellular pathways shuttle proteins from the ER for degradation: ER associated degradation (ERAD) and autophagy. Whereas the importance of ERAD in ERQC has been studied extensively and is well established, not much is known about the role of autophagy in ERQC [2].

ERAD delivers proteins from the ER for degradation by the cytoplasmic proteasome. ERAD substrates include soluble and integral-membrane proteins that fail to fold properly or assemble into complexes. Substrate recognition happens in the lumen or the membrane of the ER by chaperones (e.g., BiP). These substrates are translocated back to the cytoplasm where they are ubiquitinated and degraded by the proteasome [3,4]. Under conditions that stimulate accumulation of misfolded proteins (e.g., DTT and tunicamycin, inhibitors of disulfide-bond formation and glycosylation, respectively), ER stress and the conserved unfolded-protein response (UPR) are induced. In yeast, UPR induction requires two proteins, the endonuclease Ire1 and the transcription factor Hac1, which binds to UPR elements and stimulates the transcription of ERAD machinery components [5]. Multiple human disorders have been associated with ERAD [2].

In autophagy, cargo is delivered for degradation in the lysosome (vacuole in yeast), a major recycling cellular compartment. There are three major types of autophagy: macro, micro and chaperone mediated (CMA) [6]. Macro-autophagy, the best studied type, is a collection of cellular degradation pathways in which cargo is engulfed by a double-membrane organelle termed the autophagosome (AP) that fuses with the lysosome. All macro-autophagy pathways start with the formation of the pre-autophagosomal structure (PAS), which is mediated by the core autophagy-related proteins (Atgs). PAS includes subunits of the Atg protein complex and membranes; the latter are thought to be supplied by the only integral-membrane core Atg, Atg9 [7,8]. Macro-autophagy can be nonselective, when induced by stress, or selective, e.g., cytoplasm-to-vacuole transport (CVT), mitophagy (autophagy of mitochondria), pexophagy (autophagy of peroxisomes) [8], and ER-phagy (autophagy of the ER), which is discussed here.

Micro-autophagy and CMA, about which less is known, do not require Atgs and their cargos are not delivered through APs [6]. In micro-autophagy, cargo enters the lysosome through invagination of its membrane. For example, under certain growth conditions, peroxisome clusters can enter the lysosome via micro-pexophagy [9]. Likewise, dispensable portions of the nucleus can be delivered into the lysosome via the piecemeal micro-autophagy of the nucleus [10]. CMA, which has been described so far only in mammalian cells, is highly specific and involves translocation of unfolded proteins through the lysosomal membrane [6].

Until recently, the terms ER-phagy and reticulophagy, autophagy of the ER, have been loosely used to describe any process that delivers ER for degradation in the lysosome. For example, ER was identified as one of multiple membrane sources for AP biogenesis [11]. In addition, during prolonged ER stress, nonselective macro-autophagy can be induced [12], and was suggested to serve as a backup for ERAD [4,13]. Most notably, ER-phagy induced by ER stress [14], was recently characterized as micro-ER-phagy, which does not require the macro-autophagy machinery components [15]. Thus, almost nothing is currently known about selective macro-autophagy of the ER [16]. We have recently used the term ER-phagy to describe a Ypt1 GTPase-dependent pathway through which an overexpressed membrane protein is delivered to the vacuole for degradation [17]. The major point of that paper was to clarify that accumulation of GFP-Snc1, a marker traditionally used for endosome-to-Golgi transport, in mutant cells defective in Ypt1 function is not due to a defect in endosome-to-Golgi transport, but, rather to its role in autophagy emanating from the ER. However, under what conditions this pathway is induced, the nature of its cargos and the role of core Atgs in it, are not known. This pathway is defined here as macro-ER-phagy.

The eleven yeast Ypt GTPases and their seventy Rab homologues regulate and coordinate the multiple intra-cellular trafficking pathways [18]. These GTPases are activated by their guanine-nucleotide exchange factors (GEFs) to recruit their various effectors, which in turn mediate vesicular transport steps [19,20]. At least three Ypt GTPases were implicated in regulation of autophagy. We have shown that Ypt1, a Rab1 homolog, coordinates the first steps of two different pathways that emanate from the ER: secretion and autophagy [21,22]. Ypt1 does this in the context of two distinct GTPase modules that contain different GEFs and effectors [23]. Vps21, a Rab5 homolog, regulates two different pathways, endocytosis and autophagy, in the context of the same module [24,25]. Finally, Ypt7, a Rab7 homolog, regulates fusion of APs with the vacuole [26].

We have previously shown that components of the autophagy-specific Ypt1 module, including the Trs85-containing TRAPP III GEF, Ypt1-GTPase, and the Atg11 effector, regulate delivery of an overexpressed integral plasma membrane (PM) protein from the ER to the vacuole for degradation. The membrane protein we used was GFP-tagged Snc1-PEM; a variant of the vSNARE Snc1 that cannot be internalized by endocytosis [27]. We used the term ER-phagy for this pathway [22]. However, especially in light of recent characterization of drug-induced ER-phagy as micro-ER-phagy, it is crucial to determine if and how ER-phagy induced by overexpression of an integral-membrane protein is different from other ER-phagy processes described recently, e.g., micro-ER-phagy and backup for ERAD. Here, we characterize this pathway as macro-ER-phagy, and determine that it plays a role in vacuolar recycling of some ER-resident proteins even under normal growth conditions and can be further induced by overexpression of a single integral-membrane protein. We also show that this pathway requires core Atgs, and identify its cargos. Moreover, we define three sequential steps in this pathway, which are dependent on Atg9, Ypt1 and core Atgs, and Vps21.

Budding yeast was instrumental for current conceptual understanding of intracellular trafficking [28], autophagy [8,29], and the role of Ypt/Rab GTPases in these processes [30] in human cells. Moreover, machinery components that mediate these processes are conserved from yeast to humans and are relevant to health and disease. For example, the Ypt1 human homolog hRab1A was defined recently as an oncogene [31]. Therefore, there is no doubt that the principles of macro-ER-phagy characterized here would pertain to human cells. The potential relevance of clearance of excess membrane proteins by macro-ER-phagy to human disease is discussed.

Results

Macro-ER-phagy requires core autophagy machinery

We have previously shown that mutations in components of the autophagy-specific Ypt1 GTPase module, ypt1-1, trs85∆, and, atg11∆, are defective in delivery of overexpressed GFP-Snc1-PEM, a PM integral membrane protein, from the ER to the vacuole for degradation. We termed this pathway ER-phagy [17]. Because here we identify this pathway as macro-ER-phagy, and to distinguish it from the recently characterized micro-ER-phagy [15], we will hereinafter use the term macro-ER-phagy.

To identify other autophagy machinery components involved in macro-ER-phagy, we determined whether deletions of several Atgs result in a defect in delivering overexpressed GFP-Snc1-PEM to the vacuole through this pathway, using the following three criteria: increase in the protein level of GFP-Snc1-PEM, its accumulation in aberrant ER structures, and induction of UPR. These aberrant structures were previously identified as a cluster of ER-derived membrane-bound vesicles using immune-electron microscopy and anti-Hmg1 antibodies [17]. Their molecular composition is further characterized below.

Like Atg11, deletion of the two core autophagy components, Atg1 and Atg8, result in an increase in the GFP-Snc1-PEM protein level (Fig 1A), accumulation of aberrant Snc1-PEM intracellular structures that co-localize with the ER marker Sec61 (Fig 1B), and induction of UPR (Fig 1C). While >70% of all three mutant cells accumulate aberrant GFP-Snc1-PEM intracellular structures, the levels of protein accumulation and UPR induction vary, and are lower than those observed in ypt1-1 mutant cells (see S1A Fig). Deletion of another core autophagy component, Atg17, by itself had no effect on macro-ER-phagy. However, the double mutant atg11∆ atg17∆ displayed more severe defects in all three assays than those of the atg11∆ alone, similar to that of the ypt1-1 mutation (S1A–S1C Fig). More severe defects in other autophagy types were previously observed for the double atg11∆ atg17∆ mutant than for the single deletions [32]. These results indicate that the role of Ypt1 in macro-ER-phagy is mediated by both Atg11-dependent and-independent GTPase modules.

Atg11 plays a role in all selective autophagy processes [33]. To determine whether other selective-autophagy Atgs play a role in macro-ER-phagy, we analyzed the effect of deletions of Atg19, Atg32, and Atg36, which are required for CVT, mitophagy and pexophagy, respectively, on delivery of overexpressed GFP-Snc1-PEM to the vacuole [33]. In all three assays, atg19∆, atg32∆ and atg36∆ mutant cells behaved like wild-type cells (Fig 1D–1F). However, when combined with atg11∆, all three double mutant cells behaved like atg11∆ single mutant cells (S1D–S1F Fig).

Together, these data indicate that macro-ER-phagy requires the core autophagy machinery, but not any of the known selective autophagy components other than Atg11.

Atg9 is required for macro-ER-phagy in a step before other PAS organizers

Atg9, the only integral-membrane core Atg protein, is required for PAS formation in all autophagy processes [34]. Analysis of the atg9∆ effect on delivery of overexpressed GFP-Snc1-PEM to the vacuole revealed a phenotype different from that of mutations in other core Atgs and Ypt1 (shown in Fig 1). The level of GFP-Snc1-PEM protein was >3-fold higher than its level in WT cells, comparable to the increase in atg11∆ mutant cells (Fig 2A and 2D). A 2.5-fold increase was also observed in the cytoplasmic fluorescence of GFP-Snc1-PEM (S2A Fig). However, whereas ~75% of atg11∆ mutant cells accumulate GFP-Snc1-PEM in large intracellular structures, only 20% of the atg9∆ mutant cells contain small intracellular GFP-Snc1-PEM structures (Fig 2B and 2E), which co-localize with the ER marker Sec61-mCherry (S2A Fig). Moreover, UPR was not induced in atg9∆ mutant cells (Fig 2C and 2F). In addition, while overexpression of GFP-Snc1-PEM had no effect on the growth WT or atg11∆ mutant cells, it caused a growth defect in atg9∆ mutant cells (S2B Fig).

Two other Atgs, Atg2 and Atg18, affect the function of Atg9 by mediating its recycling from APs to peripheral sites [35]. The effects of the atg2∆ and atg18∆ mutations on macro-ER-phagy were determined. Whereas atg2∆ showed phenotypes similar to those of atg9∆, atg18∆ showed no effect (Fig 2A–2C). Different effects of Atg2 and Atg18 on autophagy were previously observed in Drosophila [36].

The different macro-ER-phagy phenotype of atg9∆ mutant cells allowed us to perform epistasis analyses with atg11∆, atg1∆, and ypt1-1. The level of GFP-Snc1-PEM in the double mutants atg9∆ atg11∆ and atg9∆ atg1∆ is similar to its level in the single mutants (Figs 2D and S2C, respectively). The fact that the level does not increase in the double mutant indicates that Atg9 functions in the same pathway as Atg11 and Atg1. Importantly, both the microscopy and the UPR assays show that the atg9∆ mutation “masks” the phenotypes of atg11∆ and atg1∆. Specifically, whereas >75% of atg11∆ and atg1∆ single-mutant cells accumulate large intracellular GFP-Snc1-PEM structures, small structures were observed only in <25% the double mutants, atg9∆ atg11∆ and atg9∆ atg1∆, similar to the phenotype of the single atg9∆ mutation (Figs 2E and S2D). Likewise, whereas UPR is induced in atg11∆ and atg1∆ single-mutant cells, it is not induced in atg9∆ atg11∆ and atg9∆ atg1∆ double mutants, similar to the phenotype of the single atg9∆ mutation (Figs 2F and S2E). Both atg9∆ and atg9∆ atg11∆ mutant cells are not defective in UPR induction, as UPR can be induced in these cells by tunicamycin (Fig 2G).

The macro-ER-phagy phenotype of the single ypt1-1 mutation is more severe than those of a single deletion of any Atg. Importantly, atg9∆ suppresses the ER-phagy phenotype of ypt1-1. First, the level of GFP-Snc1-PEM in ypt1-1 mutant cells is ~20-fold higher than its level in WT cells. In ypt1-1 atg9∆ double mutant cells, the level is reduced to ~5.5 fold (Fig 3A, left). Second, whereas 85% of ypt1-1 mutant cells contain large intracellular GFP-Snc1-PEM structures, only 28% of the ypt1-1 atg9∆ double mutant cells contain small structures, similar to atg9∆ mutant cells (Fig 3B, left). Third, the UPR is also lower in the ypt1-1 atg9∆ double mutant then in the single ypt1-1 mutant cells (Fig 3C, left).

Together, these results show that Atg9 functions upstream of Ypt1 and core Atgs in macro-ER-phagy. The fact that GFP-Snc1-PEM does not accumulate in large aberrant structures in atg9∆ mutant cells as it does in the other mutant cells, suggests that Atg9 plays a role in exit of macro-ER-phagy cargo from the ER. Interestingly, UPR induction seems to be dependent on the assembly of these structures.

One prediction from these results is that Atg9 would accumulate on aberrant ER structures in ypt1-1 mutant cells. To determine whether this is the case, WT and ypt1-1 cells expressing endogenously tagged Atg9-mCherry and overexpressing GFP-Snc1-PEM were analyzed by live-cell microscopy. Whereas in WT cells the Atg9 puncta do not co-localize with the PM-localized GFP-Snc1-PEM, in ypt1-1 mutant cells Atg9 co-localizes with the aberrant GFP-Snc1-PEM structures (Fig 3D). Thus, Atg9 is required for the formation of the aberrant ER structures in ypt1-1 mutant cells and is present on their membrane.

Characterization of macro-ER-phagy

To better characterize macro-ER-phagy, we determined whether it is affected by mutations in Sec12 and Vps4, which mediate the exocytic pathway and autophagy-independent delivery to the lysosome, respectively. Sec12 mediates ER-to-Golgi transport [37], which is also regulated by Ypt1 [38]. Because in addition to their ER-to-Golgi block, sec12ts mutant cells are also defective in ERAD at their restrictive temperature [39], it was expected that they would accumulate some GFP-Snc1-PEM in their ER even at permissive temperature. Indeed, the level of GFP-Snc1-PEM is increased ~3.5 fold when compared to WT cells, 40–50% of the cells accumulate some of it in their ER, and UPR is induced (S3 Fig). However, there are three main differences between the accumulation of GFP-Snc1-PEM in sec12-ts and ypt1-1 mutant cells. First, whereas overexpression of GFP-Snc1-PEM in ypt1-1 mutant cells results in a two-fold increase of the UPR, UPR is higher in sec12ts mutant cells that do not overexpress GFP-Snc1-PEM (S3C Fig). This result suggests that UPR induction in sec12ts mutant cells is due to a defect in ER exit of multiple proteins, and triggering the macro-ER-phagy pathway by excess GFP-Snc1-PEM might partially relieve the ER stress in these mutant cells. Second, while deletion of ATG9 masks the phenotypes of ypt1-1 (e.g., GFP-Snc1-PEM structures accumulation and UPR induction), it exacerbates those of sec12ts mutation (Fig 3A–3C, right). This result indicates that Atg9 and Sec12 do not function in the same pathway. Most importantly, we have previously shown that ypt1-1 mutant cells, regardless if they are defective in vacuolar proteolysis or not, accumulate GFP-Snc1-PEM outside their vacuole [22]. In contrast, whereas there is no GFP-Snc1-PEM in vacuoles of sec12ts mutant cells, it does accumulate in the proteolysis-defective vacuoles of sec12ts pep4∆ double-mutant cells (Fig 3E). Because GFP-Snc1-PEM can get to the vacuole only through autophagy and not through endocytosis [27], sec12ts mutant cells are not defective in autophagy. Together, these results show that unlike Ypt1, Sec12 is not required for macro-ER-phagy.

Vps4 is required for transport to the lysosome from the PM or the Golgi through late endosomes [40]. To confirm that GFP-Snc1-PEM reaches the vacuole from the ER and not from the Golgi, macro-ER-phagy of overexpressed GFP-Snc1-PEM was determined in vps4∆ mutant cells. In all three aforementioned assays, vps4∆ mutant cells behave like WT cells (S4A–S4C Fig).

Recently, a micro-ER-phagy process that does not require known autophagy machinery was described [15]. This process can be induced by adding DTT or tunicamycin to cells and observed by the presence of ER membrane “whorls” in vacuole of proteolysis-defective mutants. To confirm that the Ypt1 is not required for micro-ER-phagy, DTT-dependent formation of ER-membrane whorls was compared in YPT1 and ypt1-1 mutant cells defective in vacuolar proteolysis (pep4∆ prb1∆). Accumulation of ER whorls was similar in both strains (S4D Fig), indicating that Ypt1 does not play a role in micro-ER-phagy. Therefore, GFP-Snc1-PEM accumulation in ypt1-1 mutant cells is caused by a defect in a process distinct from micro-ER-phagy.

Our finding that the core autophagy machinery is required for delivery of GFP-Snc1-PEM to the vacuole supports the idea that the process described here is macro-ER-phagy. To confirm this idea, we determined whether yDsRed-Snc1-PEM passes through APs en route to the vacuole. Recently, we showed that the Rab5 homolog Vps21 plays a role in autophagy as vps21∆ mutant cells accumulate AP structures marked by Atg8 under starvation [24]. This indicates that autophagy is blocked in vps21∆ mutant cells after the formation of APs. Under normal growth conditions, both WT and vps21∆ mutant cells have one Atg8 dot per cell, representing the phagophore or AP [41]. Co-localization of overexpressed yDsRed-Snc1-PEM with yEGFP-Atg8 was observed in ~70% of vps21∆ mutant cells, as compared to ~4% of WT cells (Fig 4A, top). This vps21∆ phenotype is also different from that of the ypt1-1 single mutation and the ypt1-1 vps21∆ double mutation. As we have previously shown, ypt1-1 mutant cells are defective in PAS formation and contain multiple Atg8 dots per cell [22,42]. Even though yDsRed-Snc1-PEM accumulates in ypt1-1 single- and ypt1-1 vps21∆ double-mutant cells, it does not co-localize with the multiple Atg8 dots (Fig 4A, bottom). The finding that the ypt1-1 mutation overrides the vps21∆ phenotype indicates that Ypt1 and Vps21 function sequentially in the same pathway. Moreover, the co-localization of yDsRed-Snc1-PEM with Atg8 in vps21∆ mutant cells indicates that it passes through APs en route to the vacuole and confirms that this pathway is macro-ER-phagy.

UPR plays a role in ERAD [5] and is induced when macro-ER-phagy is blocked [17]. To determine whether UPR is required for macro-ER-phagy, accumulation of overexpressed GFP-Snc1-PEM was tested in ire1∆ and hac1∆ mutant cells, which are defective in UPR induction. In WT (YPT1) cells, deletion of either IRE1 or HAC1 abolishes UPR, but does not result in accumulation of GFP-Snc1-PEM (Fig 4B–4D, left). In ypt1-1 mutant cells, deletion of either IRE1 or HAC1 abolishes UPR induction, but does not affect accumulation of GFP-Snc1-PEM (Fig 4B–4D, right). These results show that Ire1/Hac1-dependent UPR induction is not required for macro-ER-phagy and does not affect the formation of aberrant ER structures in ypt1-1 mutant cells.

Macro-ER-phagy cargos

We have shown that overexpressed GFP-Snc1-PEM, a chimeric integral PM protein, can serve as a cargo for macro-ER-phagy [17]. We wished to identify a native protein that when overexpressed would be a cargo for ER-phagy. For this purpose we chose the PM multi-drug transporter Snq2 that contains multiple trans-membrane domains. Snq2 tagged at its C-terminus with yEGFP was overexpressed from a 2μ plasmid in WT and ypt1-1 mutant cells. In ypt1-1 mutant cells, like GFP-Snc1-PEM, the level of Snq2-yEGFP was increased (by ~10-fold), it accumulated in aberrant ER structures in 80% of the cells, and UPR was induced (Fig 5A–5C). Thus, overexpressed Snq2-yEGFP, like GFP-Snc1-PEM, is transported through macro-ER-phagy for degradation in the vacuole.

To study the effect of overexpression of multiple membrane proteins on macro-ER-phagy, Snq2-yEGFP and DsRed-Snc1-PEM were co-overexpressed in WT and atg11∆ mutant cells. In WT cells, while overexpression of a single membrane protein did not have any effect, overexpression of two proteins resulted in some increase in the protein levels, and they co-localized in the vacuole of ~12% of the cells (Figs 5D and 5E and S5A). In atg11∆ mutant cells, while overexpression of a single membrane protein had a moderate phenotype, overexpression of the two resulted in a synergistic effect. The protein levels were increased >25 fold over that of WT cells (single protein levels were increased 2.5–6 fold) (Fig 5D). In 87% of the cells the two proteins co-localized in aberrant intra-cellular structures (compared to 50–65% for single protein) (Figs 5E and S5A). Moreover, the fluorescence of these intra-cellular structures was 5-fold brighter than that of each single protein (Fig 5F). Finally, whereas overexpression of a single membrane protein did not affect the growth of atg11∆ mutant cells, overexpression of two caused a growth defect (S5B Fig). These results suggest that when macro-ER-phagy is partially defective, as in atg11∆ mutant cells, the extra burden of overexpression of multiple membrane proteins can be detrimental.

Together, these results indicate that excess of integral membrane proteins, such as Snc1-PEM and Snq2, are transported to the vacuole through macro-ER-phagy.

ER-resident proteins in macro-ER-phagy

We wished to determine which ER-resident proteins are transported together with GFP-Snc1-PEM to the vacuole via the macro-ER-phagy pathway. Five ER-resident proteins were used for this analysis: three integral membrane proteins, Sec61 (translocon subunit), Hmg1 (sterol biogenesis), and Sec12 (ER-exit sites), a cytoplasmic coat protein, Sec13 (ER exit sites), and an ER-lumen chaperon, Kar2 (BiP) (Fig 6A). Protein accumulation was determined by immunoblot and microscopy analyses in WT (YPT1) and ypt1-1 mutant cells in which vacuolar proteolysis was normal (PEP4 PRB1) or defective (pep4∆ prb1∆). The analysis was done without and with over-expression of GFP-Snc1-PEM.

We have previously shown that some resident-ER proteins accumulate in ypt1-1 mutant cells when compared to WT cells even without overexpression of GFP-Snc1-PEM. This phenotype was observed using live-cell microscopy and tagged resident ER proteins, e.g., Hmg1 and Sec61 [17]. Here, the microscopy analysis is supported by an immunoblot analysis. The levels of Sec61 and Hmg1 were determined in cells that do not overexpress GFP-Snc1-PEM. In YPT1 (WT) cells defective in vacuolar proteolysis (pep4∆ prb1∆), the levels of Sec61 and Hmg1 were increased by 1.2–1.6 fold. In ypt1-1, regardless if the vacuolar proteolysis is normal or defective, the levels of Sec61 and Hmg1 were increased by ~2–2.6 fold (Figs 6B and 6D and S6A). In contrast, the levels of Sec12, Sec13 and Kar2 were not increased in these mutant cells (Figs 6C and 6D and S6B and S6C). Therefore, ~20–40% of some ER resident proteins, but not all, are shuttled to the vacuole for degradation in WT and ypt1-1 mutant cells even in cells that do not over-express GFP-Snc1-PEM.

The previously observed Hmg1 and Sec61 accumulation in ypt1-1 mutant cells was exacerbated when GFP-Snc1-PEM was overexpressed. In addition, we have previously shown that in YPT1 (WT) cells GFP-Snc1-PEM is transported to the vacuole for degradation [17]. We wished to determine whether ER-resident proteins are shuttled to the vacuole for degradation with over-expressed GFP-Snc1-PEM.

Hmg1 and Sec61 behaved like Snc1-PEM in wild type cells that overexpress GFP-Snc1-PEM. Immuno-blot analysis shows that their levels increased by 10 fold in cells defective in vacuolar proteolysis, indicating that they were delivered to the vacuole (Figs 6F and 6H and S6F). The degradation rate of Sec61 was compared in WT cells, which either over-express GFP-Snc1-PEM or not, using cycloheximide to inhibit protein translation. The half-life of Sec61 is shortened by 7 fold in cells over-expressing GFP-Snc1-PEM (from 29 to 4 hours; S6D Fig). The finding that the stability of the ER resident protein Sec61 is reduced in WT cells over-expressing GFP-Snc1-PEM further supports the idea that it is shuttled to the vacuole for degradation under these conditions. However, in spite of the fact that ~95% of Sec61 and Hmg1 were degraded in cells overexpressing GFP-Snc1-PEM, their steady-state level did not change (Figs 6E and S6E). In contrast to Sec61 and Hmg1, the levels of Sec12, Sec13 and Kar2 in vacuolar proteolysis defective cells overexpressing GFP-Snc1-PEM were very slightly changed (1.5–2 fold) (Figs 6G and 6H and S6G–S6H, left). Thus, in WT cells, >90% of some, but not all, ER resident proteins are shuttled for degradation in the vacuole with overexpressed GFP-Snc1-PEM.

In ypt1-1 mutant cells that overexpress GFP-Snc1-PEM, the levels of Hmg1 and Sec61, like that of Snc1-PEM, were increased by ~15 fold regardless whether vacuolar proteolysis was normal or defective, indicating that they accumulate before reaching the vacuole. In contrast, the levels Sec12, Sec13 and Kar2 increased by 4, 3, and 2 fold, respectively (Figs 6F–6H and S6F–S6H, right).

In the microscopy analysis, Hmg1 and Sec61 also behaved like GFP-Snc1-PEM. In YPT1 (WT) cells defective in vacuolar proteolysis, 60–70% of the intra-cellular GFP-Snc1-PEM co-localized with mCherry-tagged Hmg1 and Sec61 (Figs 7A and 7D and S7A). The proteins probably co-localize in the vacuole because co-localization is observed only in cells defective in vacuolar proteolysis and, using a vacuolar membrane stain, we have previously shown that GFP-Snc1-PEM accumulates inside the vacuole of pep4∆ cells [17]. This point was confirmed for Hmg1, which co-localizes with Snc1-PEM in the vacuole, using three-color fluorescence microscopy (Fig 7B). In contrast, very little co-localization was observed for Sec13, Sec12 and Kar2 (Figs 7A and 7D and S7A). In ypt1-1 mutant cells, >70% of GFP-Snc1-PEM co-localized with Hmg1 and Sec61, regardless of the vacuolar proteolysis state. For Sec12, Sec13 and Kar2, the levels were lower: 38, 14 and 12%, respectively (Figs 7C and 7D and S7B).

These results show that ~20–40% of some, but not all, ER-resident proteins are transported to the vacuole through macro-ER-phagy in WT cells. In cells over-expressing excess integral membrane proteins such as Snc1-PEM, >90% of these ER proteins are corralled to this pathway. Interestingly, even though the ER resident protein Sec61 is shuttled to the vacuole for the degradation when GFP-Snc1-PEM is overexpressed, its steady state level does not change.

Discussion

Results presented here define a novel ER quality-control pathway, macro-ER-phagy. This pathway delivers excess of integral-membrane proteins from the ER to the lysosome for degradation and requires core autophagy machinery and at least two Ypt GTPases, Ypt1 and Vps21 (Ypt51). It was defined as macro-ER-phagy based on the requirement of core Atgs and the accumulation of the cargo in Atg8-marked autophagic structures (phagophore and APs) in vps21∆ mutant cells. Macro-ER-phagy is different from nonselective autophagy because it does not require starvation or prolonged ER stress, and from other specific autophagy processes, because it does not require known specific Atgs (like Atg19, Atg32, and Atg36). It is also different from micro-ER-phagy, which does not require Atgs [15] or Ypt1 GTPase (shown here). Finally, macro-ER-phagy is different from the other ERQC pathway ERAD in which proteins are extracted from the ER lumen through the membrane and delivered to the proteasome for degradation (Fig 8A). When macro-ER-phagy is impaired, UPR is induced, indicating that accumulation of its cargo causes ER stress. However, UPR induction is not required for macro-ER-phagy.

Macro-ER-phagy cargo

The cargo shuttled through the macro-ER-phagy pathways is different from cargo delivered by other known ERQC pathways. Whereas the cargo of the ERAD and micro-ER-phagy pathway are misfolded proteins (recognized in the ER lumen or in the ER membrane) and extra membrane, respectively [3,15], the cargo of the macro-ER-phagy is excess of integral-membrane proteins. Based on the fact that one such cargo, GFP-Snc1-PEM, does not have a lumenal domain, we propose that the macro-ER-phagy cargo is recognized in the cytoplasm.

What fraction of integral-membrane proteins is transported through basal macro-ER-phagy? Without overexpression of a membrane protein, about 20–50% of the ER resident proteins Sec61 and Hmg1 are transported to the vacuole for degradation. This estimate is based on a 1.2–1.6 fold increase in cells defective in vacuolar proteolysis, and on the 2–2.5 fold accumulation in ypt1-1 mutant cells regardless of whether vacuolar proteolysis is normal or not. This estimate is within the 30–75% range previously reported for ER proteins that do not pass the ERQC [1].

Importantly, cells that overexpress the integral-membrane protein GFP-Snc1-PEM, shuttle ~95% of this protein to the vacuole for degradation via macro-ER-phagy. This fraction is estimated based on the following two results: ~20-fold increase in GFP-Snc1-PEM level in cells defective in macro-ER-phagy (e.g., ypt1-1), and >10-fold increase in cells defective in vacuolar proteolysis (pep4∆ prb1∆). Moreover, >90% of some ER-resident membrane proteins (e.g., Sec61 and Hmg1) are also transported through macro-ER-phagy for degradation in cells overexpressing GFP-Snc1-PEM. In spite of this increase in the degradation of ER resident proteins upon over-expression of a single integral membrane protein, the steady state level of Sec61 and Hmg1 remained unchanged.

We propose that macro-ER-phagy is a “housekeeping” pathway that delivers excess resident-ER membrane proteins to the lysosome for degradation. When an integral membrane protein is overexpressed, ~95% of this protein is shuttled through this pathway together with >90% of the ER that contain certain resident-ER membrane proteins. Therefore, macro-ER-phagy of some resident ER proteins is induced from 20–50% to >90% by over-expression of a single membrane protein. The observation that the steady state level did not plunge under these conditions suggests that cells regulate the level of these ER resident proteins.

Delineation of three distinct steps of the macro-ER-phagy pathway

Based on data presented here, we delineate three sequential steps of the macro-ER-phagy pathway: In the first Atg9-dependent step, membranes containing macro-ER-phagy cargo form ER-to-autophagy membranes (ERAM). If this step is blocked, cargo accumulates in the ER, but UPR is not induced. In the second step, which is dependent on Ypt1, ERAM and the core Atgs form the PAS. If this step is blocked, ERAM accumulate and UPR is induced, even though UPR induction is not required for macro-ER-phagy. In the third Atg8- and Vps21-mediated step, APs are formed and fuse with the lysosome (see model in Fig 8B).

Atg9, the only integral-membrane core Atg, was implicated in delivering membrane to PAS [7,8]. However, until now its function could not be separated from that of the other PAS organizers. Because the macro-ER-phagy phenotype of atg9∆ is different from, and can mask, the phenotypes of ypt1-1, atg1∆ and atg11∆, the two steps could be separated and their order could be defined. This is the first time that the function of Atg9 is uncoupled from that of the other core Atgs in any autophagy process. In macro-ER-phagy, Atg9 is required for ERAM formation and is present on these membranes. Interestingly, in the absence of over-expressed cargo, a large portion of peripheral Atg9 structures co-localize with ER [43].

Ypt1 regulates ER-to-Golgi transport and all types of macro-autophagy in the context of different GEF-GTPase-effector modules [23]. Even though we established the ypt1-1 mutation affects autophagy and not ER-to-Golgi transport [21,22], it was important to ensure that mutations in other ER-to-Golgi and Golgi-to-vacuole regulators do not exhibit macro-ER-phagy phenotypes. Sec12 mediates exit from the ER towards the Golgi. In sec12-ts mutant cells, some membrane proteins are trapped in the ER, and ER stress is high. However, these mutant cells are not defective in macro-ER-phagy because excess membrane proteins can reach the vacuole, and atg9∆ enhances their phenotype rather than masking it. In addition, inhibition of transport from the Golgi or endosomes in vps4∆ mutant cells did not affect macro-ER-phagy. Moreover, even though the macro-ER-phagy phenotype of ypt1-1 is more severe than deletion of any single Atg, the observation that the phenotype of the atg11∆ atg17 double mutant is similar to that of ypt1-1 suggests that the ypt1-1 phenotype is caused by a defect in autophagy. All these results support the idea that the function of Ypt1 in macro-ER-phagy is unrelated to its function in ER-to-Golgi transport.

Vps21 was used here to show that macro-ER-phagy cargo utilizes APs like other macro-autophagy pathways. We have recently shown that Vps21 is required for specific and nonspecific autophagy, and vps21∆ mutant cells accumulate autophagic structures marked by Atg8 [24]. Here we show that Vps21 also plays a role in macro-ER-phagy because a macro-ER-phagy cargo accumulates in Atg8-marked autophagic structures of vps21∆ mutant cells. The observation that this phenotype can be masked by ypt1-1 supports the idea that Ypt1 functions before Vps21 in this pathway.

While UPR is induced when the second step of macro-ER-phagy is blocked, it is not required for this process. This idea is based on the observation that deletion of IRE1 or HAC1, which completely abolish the UPR, does not affect macro-ER-phagy in YPT1 (WT) cells, and can uncouple UPR from the formation of ERAM in ypt1-1 mutant cells. While it is not clear why UPR is induced in mutant cells defective in the second step of macro-ER-phagy (e.g., ypt1-1, atg1∆, atg8∆ and atg11∆), the finding that Kar2 (BiP) is excluded from ERAM in ypt1-1 mutant cells supports the model that sequestration of Kar2 from Ire1 (which presumably is retained) is one of the requirements for UPR induction [44].

Open questions

While overexpression of integral PM proteins was used here to detect clear defects in the pathway, partial defects in delivery of some ER-resident proteins to the vacuole were observed without it in ypt1-1 mutant cells [17]. Moreover, we show here that some resident ER proteins are delivered to the vacuole through basal macro-ER-phagy also in wild type cells, and overexpression of a single integral PM protein further induces this pathway. This suggests that macro-ER-phagy is a constitutive ERQC process that clears excess membrane proteins from the ER. Our results also imply the existence of a complementary process that ensures that the steady state level of certain resident ER proteins is maintained when macro-ER-phagy is induced by over-expression of membrane proteins. While it is not clear whether this steady state level is regulated at the level of mRNA or protein, it seems that it does not involve UPR, which regulates the expression of multiple genes [45], because UPR is not induced in wild type cells overexpressing integral-membrane proteins (Figs 5C and S3C). Specific mechanisms that underlie basal and induced macro-ER-phagy and the regulation of ER-resident proteins level when macro-ER-phagy is induced, need to be deciphered.

Other open questions to be addressed in the future are how macro-ER-phagy cargo is recognized and sorted to ERAM and the relationship between this pathway and ERAD. We propose that macro-ER-phagy cargo is recognized either on the cytoplasmic side of the ER or in the ER membrane, because at least one cargo, GFP-Snc1-PEM, does not have a lumenal domain. Under ER stress, the ERAD-M and ERAD-C pathways recognize misfolded intra-membrane and cytosolic domains of membrane proteins, respectively [46]. While it is possible that excess integral-membrane proteins, especially when tagged with a fluorescence moiety, are partially misfolded, it seems that ERAD does not play a major role in their clearance because deletion of Ire1 and Hac1, which are required for ERAD induction, does not affect this clearance. To determine whether ERAD plays a role in the degradation of excess ER membrane proteins, the effect of ERAD mutations, alone and in combination with autophagy mutations, on this process should be determined in future experiments. It is intriguing if and how proteins are sorted between macro-ER-phagy and ERAD for degradation, whether they are sequestered to different domains, and whether yet unknown ER-phagy-specific Atgs play a role in this process. In addition, cooperation between ER-phagy and other ERQC processes has been proposed [4]. The idea is that each pathway can serve as a backup for the other. The synergistic effect of overexpression of multiple membrane proteins in atg11∆ mutant cells supports this idea. We propose that these mutant cells cope with excess of a single protein using another ERQC pathway, possibly ERAD, while overexpression of two proteins saturates this outlet.

Another question is whether ER-exit sites towards the Golgi (ERES) and ER-phagy overlap. Recently, ERES were proposed to be the site of AP biogenesis [47]. Our result that Sec12, an ERES integral-membrane protein, is not delivered to the vacuole through macro-ER-phagy, does not support a role for ERES in this process.

What is the relevance of degradation of excess membrane proteins by macro-ER-phagy? First, specific impairment of macro-ER-phagy should be considered in studies that require overexpression of membrane proteins. For example, ERQC was identified as the bottleneck of overexpression of heterologous proteins in yeast. Co-overexpression of chaperons, like BiP, did not affect this block [48], supporting the idea that macro-ER-phagy, and not ERAD, is the likely cause of this bottleneck. Most importantly, overexpression of membrane proteins has been associated with multiple human diseases. For example, amplification of HER2, a human epidermal growth factor receptor, occurs in ~20% of breast cancers and is associated with a more aggressive disease [49,50]. Likewise, overexpression of the M oncostatin receptor is associated with increased aggressiveness of cervical cancer and is considered a therapeutic target [51]. Moreover, overexpression of the P-glycogen efflux pump is associated with bone inflammation and is considered a potential therapeutic target [52]. Finally, chronic ER stress is associated with neurodegenerative diseases and therapeutic benefits of chemical autophagy activators were reported [53]. Therefore, if the mechanism we unraveled here is conserved from yeast to human like all other basic cellular processes, macro-ER-phagy of excess membrane protein would be relevant to human disease.

Note added in proof

While this paper was under revision, Mochida et al [54] reported the identification of two new receptors, Atg39 and Atg40, required for autophagy of the ER during nitrogen starvation, which induces non-selective autophagy. Interestingly, Atg40 is similar to the FM134B reticulon, an autophagy receptor of ER in mammals, which was implicated in neuropathy in humans [55]. Future studies should clarify whether these new receptors play a role in macro-ER-phagy of ER proteins under normal growth conditions described here.

Materials and Methods

Strains, plasmids, construction and reagents

Details about strains, plasmids and reagents used in this manuscript are given in Supplementary Experimental Procedures (S1 File), and S1 and S2 Tables. Construction of strains and plasmids is described in Supplementary Experimental Procedures (S1 File).

Yeast culture conditions

All yeast strains were grown in rich (YPD) or minimal (SD) media containing the necessary amino acid supplements. In tunicamycin experiments, cells were incubated with 5 μg/ml tunicamycin for 1.5 hours before collecting them. Experiments involving DTT treatment were performed as previously described [15].

Protein level analysis

The protein level of GFP-Snc1-PEM, HA-tagged Sec61, Sec13 and Sec12 was determined as previously described [17]. The protein level of mCherry-tagged Hmg1 and Kar2 was determined according to the previously described methods [56,57]. Quantification was done using ImageJ and adjusted to the loading control.

Microscopy

For live-cell microscopy, cells expressing fluorescently tagged proteins were grown to mid-log phase in appropriate media. Fluorescence microscopy was performed using deconvolution Axioscope microscope (Carl Zeiss, Thornwood, NY) with FITC (GFP, yEGFP) and TexasRed (mCherry) sets of filters. Labeling of vacuole membranes with FM4-64 was done using a 5 min pulse followed by 60 min chase as previously described [58]. Labeling the vacuolar lumen with CMAC-Arg was done according to the manufacturer’s instructions. Immunofluorescence microscopy of yeast cells was performed as previously described [59].

UPR β-gal assay

The β-gal assay was done with cells as previously described [17]. Briefly, cells were transformed with a plasmid (pJC104) expressing the LacZ gene behind four UPR elements [60]. The level of β-galactosidase in cell extracts was determined as β-gal (Miller) units [61]. The values are 0.6–1.0 units for WT cells. Graphs represent percent of UPR in experimental cells (e.g., mutant cells) as fold of β-Gal units in WT cells; averages and error bars (represent STDEV) were calculated from two independent reactions; each result represents at least two different experiments from independent transformants. Statistical significance (p value) was calculated from two different experiments using independent transformants, each with two independent reactions (total of four reactions). In our experiments, tunicamycin treatment (5 μg/ml for 90 min) results in a 12-22-fold induction over untreated WT cells.

Supporting Information

Attachment 1

Attachment 2

Attachment 3

Attachment 4

Attachment 5

Attachment 6

Attachment 7

Attachment 8

Attachment 9

Attachment 10