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A New Autophagy-related Checkpoint in the Degradation of an ERAD-M Target

  • Edith Kario
    Affiliations
    Departments of Biological Regulation
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  • Nira Amar
    Affiliations
    Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
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  • Zvulun Elazar
    Correspondence
    Supported in part by the Israel Science Foundation, the Israeli Cancer Research Foundation, and the Weizmann Institute Minerva Center. Incumbent of the Harold Korda Chair of Biology. To whom correspondence may be addressed. Tel.: 972-8-9343682; Fax: 972-8-9344112
    Affiliations
    Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
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  • Ami Navon
    Correspondence
    Incumbent of the Recanati career development chair of cancer research. Research in the laboratory of A. N. is supported by the Israel Science Foundation, the Minerva Foundation (Germany), the German-Israeli Foundation for Scientific Research and Development, and a special gift from Rolando Uziel. To whom correspondence may be addressed. Tel.: 972-8-9343719; Fax: 972-8-9344116
    Affiliations
    Departments of Biological Regulation
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  • Author Footnotes
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S11.
Open AccessPublished:January 12, 2011DOI:https://doi.org/10.1074/jbc.M110.177618
      The endoplasmic reticulum (ER) harbors elaborate quality control mechanisms to ensure proper folding and post-translational modifications of polypeptides targeted to this organelle. Once an aberrant protein is detected, it is dislocated from the ER and routed to the proteasome for destruction. Autophagy has been recently implicated in the elevation of the ER stress response; however, the involvement of this pathway in selective removal of ER-associated degradation (ERAD) substrates has not been demonstrated. In the present study, we show that an ER membrane lesion, associated with the accumulation of the yeast ERAD-M substrate 6Myc-Hmg2p elicits the recruitment of Atg8 and elements of the cytosol to vacuole targeting (CVT) to the membrane, leading to attenuation in the degradation process. Deletion of peptide:N-glycanase (PNG1) stabilizes this association, a process accompanied by slowdown of 6Myc-Hmg2p degradation. Truncation of the unstructured C-terminal 23 amino acids of 6Myc-Hmg2p rendered its degradation PNG1-independent and allowed its partial delivery to the vacuole in an autophagy-dependent manner. These findings demonstrate a new conduit for the selective vacuolar/lysosomal removal of ERAD misfolded proteins by an autophagy-related machinery acting concomitantly with the proteasome.

      Introduction

      The endoplasmic reticulum (ER)
      The abbreviations used are: ER
      endoplasmic reticulum
      ERAD
      ER-associated degradation
      IP
      immunoprecipitation.
      is the port of entry into the secretory pathway. A fraction of newly synthesized polypeptides remains in the ER to serve in various metabolic and structural functions. Almost all proteins processed in the ER acquire co- or post-translational modifications, of which the most common and diverse is glycosylation (
      • Helenius A.
      • Aebi M.
      ). Correctly folded proteins that pass the ER quality control checkpoints continue to their final destination, whereas misfolded proteins are retained in the ER and subsequently eliminated. Their removal entails dislocation across the ER membrane, ubiquitination, extraction into the cytosol, and proteasomal degradation (
      • Vembar S.S.
      • Brodsky J.L.
      ). Protein breakdown by ER-associated degradation (ERAD) is not a uniform process. ERAD substrates are divided into three classes, depending on the location of the lesion within the aberrant protein; ERAD-L substrates are either soluble or membrane proteins with a luminal lesion, whereas ERAD-M and ERAD-C substrates are transmembrane proteins with a misfolded transmembrane or cytosolic domain, respectively (
      • Carvalho P.
      • Goder V.
      • Rapoport T.A.
      ,
      • Vashist S.
      • Ng D.T.
      ).
      Degradation of all ERAD substrates begins with recognition of the misfolded polypeptides within the ER by chaperones, which detect exposed hydrophobic patches of misfolded proteins, aberrant disulfide bonds, or unassembled protein complexes (
      • Vembar S.S.
      • Brodsky J.L.
      ). N-Linked glycans have a key role in quality control and in the recognition of improperly folded proteins. Within the ER, glycans are recognized by lectins, such as the yeast Htm1/Mnl1 and Yos9 and their mammalian orthologs EDEM and EDEM2 (
      • Mast S.W.
      • Diekman K.
      • Karaveg K.
      • Davis A.
      • Sifers R.N.
      • Moremen K.W.
      ,
      • Hosokawa N.
      • Wada I.
      • Hasegawa K.
      • Yorihuzi T.
      • Tremblay L.O.
      • Herscovics A.
      • Nagata K.
      ,
      • Olivari S.
      • Galli C.
      • Alanen H.
      • Ruddock L.
      • Molinari M.
      ,
      • Nakatsukasa K.
      • Nishikawa S.
      • Hosokawa N.
      • Nagata K.
      • Endo T.
      ,
      • Bhamidipati A.
      • Denic V.
      • Quan E.M.
      • Weissman J.S.
      ,
      • Jakob C.A.
      • Bodmer D.
      • Spirig U.
      • Battig P.
      • Marcil A.
      • Dignard D.
      • Bergeron J.J.
      • Thomas D.Y.
      • Aebi M.
      ,
      • Oda Y.
      • Hosokawa N.
      • Wada I.
      • Nagata K.
      ). Glycans may also be essential for retrotranslocation of ER-misfolded glycoproteins to the cytosol. For example, removal of the terminal glycan from the ERAD substrate CPY* (mutated yeast vacuolar carboxyl peptidase Y), which is decorated with four N-linked glycans, results in inhibition of its degradation and its consequent accumulation in the ER (
      • Spear E.D.
      • Ng D.T.
      ,
      • Kostova Z.
      • Wolf D.H.
      ). Moreover, certain E3 ubiquitin ligases (Fbs1 and Fbs2) bind glycans of misfolded glycoproteins in the cytosol and promote their ubiquitination and proteasomal degradation (
      • Yoshida Y.
      • Tokunaga F.
      • Chiba T.
      • Iwai K.
      • Tanaka K.
      • Tai T.
      ,
      • Yoshida Y.
      • Chiba T.
      • Tokunaga F.
      • Kawasaki H.
      • Iwai K.
      • Suzuki T.
      • Ito Y.
      • Matsuoka K.
      • Yoshida M.
      • Tanaka K.
      • Tai T.
      ).
      ER stress ensues when the amount of client proteins that emerges into the ER exceeds its overall folding capacity or upon overload of the ER with damaged or misfolded polypeptides. This challenge activates an intricate cytoprotective ER-to-nucleus signaling cascade, collectively termed the unfolded protein response, which in turn up-regulates the ER capacity to handle the load of misfolded proteins (
      • Cox J.S.
      • Walter P.
      , ,
      • Sidrauski C.
      • Cox J.S.
      • Walter P.
      ). Another potential cellular route for clearing ER-misfolded polypeptides that exceed the capacity of the ERAD machinery may involve routing of these misfolded proteins to the vacuole/lysosome. In fact, previous studies have demonstrated that upon overexpression of CPY*, a portion of this ERAD-L substrate is transported to the vacuole for degradation (
      • Spear E.D.
      • Ng D.T.
      ).
      Autophagy may also serve to deliver some of the aberrant proteins for vacuolar/lysosomal removal. This pathway, engaged in the removal of superfluous or damaged organelles and cytosolic proteins, was found important for development, cellular maintenance, and remodeling (
      • Levine B.
      • Klionsky D.J.
      ,
      • Meijer A.J.
      • Codogno P.
      ). The yeast ubiquitin-like protein Atg8 and its mammalian orthologs LC-3, GATE-16, and GABARAP serve as key components of autophagy (
      • Kirisako T.
      • Baba M.
      • Ishihara N.
      • Miyazawa K.
      • Ohsumi M.
      • Yoshimori T.
      • Noda T.
      • Ohsumi Y.
      ,
      • Kabeya Y.
      • Mizushima N.
      • Yamamoto A.
      • Oshitani-Okamoto S.
      • Ohsumi Y.
      • Yoshimori T.
      ,
      • Weidberg H.
      • Shvets E.
      • Shpilka T.
      • Shimron F.
      • Shinder V.
      • Elazar Z.
      ). In the early stages of autophagosome formation, Atg8 becomes lipidated and accumulates in preautophagosomal structures, which are the nucleation sites for autophagosomes (
      • Suzuki K.
      • Kirisako T.
      • Kamada Y.
      • Mizushima N.
      • Noda T.
      • Ohsumi Y.
      ). Atg8 remains attached to the autophagosome and is delivered to the vacuole (
      • Kirisako T.
      • Baba M.
      • Ishihara N.
      • Miyazawa K.
      • Ohsumi M.
      • Yoshimori T.
      • Noda T.
      • Ohsumi Y.
      ,
      • Huang W.P.
      • Scott S.V.
      • Kim J.
      • Klionsky D.J.
      ,
      • Ichimura Y.
      • Kirisako T.
      • Takao T.
      • Satomi Y.
      • Shimonishi Y.
      • Ishihara N.
      • Mizushima N.
      • Tanida I.
      • Kominami E.
      • Ohsumi M.
      • Noda T.
      • Ohsumi Y.
      ,
      • Kirisako T.
      • Ichimura Y.
      • Okada H.
      • Kabeya Y.
      • Mizushima N.
      • Yoshimori T.
      • Ohsumi M.
      • Takao T.
      • Noda T.
      • Ohsumi Y.
      ). Autophagy is normally elicited under stress conditions, such as nutrient or growth factor deprivation, and fulfills a protective role in human diseases, including cancer, some types of neurodegenerative diseases, and muscular disorders (
      • Shintani T.
      • Klionsky D.J.
      ,
      • Berger Z.
      • Ravikumar B.
      • Menzies F.M.
      • Oroz L.G.
      • Underwood B.R.
      • Pangalos M.N.
      • Schmitt I.
      • Wullner U.
      • Evert B.O.
      • O'Kane C.J.
      • Rubinsztein D.C.
      ). A typical scenario for such conditions may involve aggregation of misfolded proteins (i.e. polyglutamin, mutants of ataxin3, Huntingtin, mutant α-synuclein, and different forms of Tau) that escape proteasomal degradation. In such circumstances, macroautophagy may replace the proteasome and become the major clearance pathway (
      • Berger Z.
      • Ravikumar B.
      • Menzies F.M.
      • Oroz L.G.
      • Underwood B.R.
      • Pangalos M.N.
      • Schmitt I.
      • Wullner U.
      • Evert B.O.
      • O'Kane C.J.
      • Rubinsztein D.C.
      ,
      • Kaganovich D.
      • Kopito R.
      • Frydman J.
      ,
      • Webb J.L.
      • Ravikumar B.
      • Atkins J.
      • Skepper J.N.
      • Rubinsztein D.C.
      ,
      • Shibata M.
      • Lu T.
      • Furuya T.
      • Degterev A.
      • Mizushima N.
      • Yoshimori T.
      • MacDonald M.
      • Yankner B.
      • Yuan J.
      ,
      • Iwata A.
      • Riley B.E.
      • Johnston J.A.
      • Kopito R.R.
      ,
      • Qin Z.H.
      • Wang Y.
      • Kegel K.B.
      • Kazantsev A.
      • Apostol B.L.
      • Thompson L.M.
      • Yoder J.
      • Aronin N.
      • DiFiglia M.
      ,
      • Ravikumar B.
      • Vacher C.
      • Berger Z.
      • Davies J.E.
      • Luo S.
      • Oroz L.G.
      • Scaravilli F.
      • Easton D.F.
      • Duden R.
      • O'Kane C.J.
      • Rubinsztein D.C.
      ). Shuttle proteins, such as P62/SQSTM1 and HDAC6, potentially serve in recruiting polyubiquitinated aggregated proteins into autophagic vesicles (
      • Iwata A.
      • Riley B.E.
      • Johnston J.A.
      • Kopito R.R.
      ,
      • Bjørkøy G.
      • Lamark T.
      • Brech A.
      • Outzen H.
      • Perander M.
      • Overvatn A.
      • Stenmark H.
      • Johansen T.
      ,
      • Pankiv S.
      • Clausen T.H.
      • Lamark T.
      • Brech A.
      • Bruun J.A.
      • Outzen H.
      • Øvervatn A.
      • Bjørkøy G.
      • Johansen T.
      ,
      • Zatloukal K.
      • Stumptner C.
      • Fuchsbichler A.
      • Heid H.
      • Schnoelzer M.
      • Kenner L.
      • Kleinert R.
      • Prinz M.
      • Aguzzi A.
      • Denk H.
      ,
      • Nagaoka U.
      • Kim K.
      • Jana N.R.
      • Doi H.
      • Maruyama M.
      • Mitsui K.
      • Oyama F.
      • Nukina N.
      ,
      • Pandey U.B.
      • Nie Z.
      • Batlevi Y.
      • McCray B.A.
      • Ritson G.P.
      • Nedelsky N.B.
      • Schwartz S.L.
      • DiProspero N.A.
      • Knight M.A.
      • Schuldiner O.
      • Padmanabhan R.
      • Hild M.
      • Berry D.L.
      • Garza D.
      • Hubbert C.C.
      • Yao T.P.
      • Baehrecke E.H.
      • Taylor J.P.
      ,
      • Kawaguchi Y.
      • Kovacs J.J.
      • McLaurin A.
      • Vance J.M.
      • Ito A.
      • Yao T.P.
      ). Autophagy has also been implicated in ER quality control, providing an alternative mechanism for the clearance of misfolded proteins that accumulate in the ER lumen. For instance, the Z variant of human α-1 antitrypsin (ATZ), a protease inhibitor produced in the liver, may misfold and accumulate in the ER, causing liver disease. Recent studies have indicated that degradation of the misfolded ATZ involves both the general ER quality control and the autophagic systems (
      • Teckman J.H.
      • Perlmutter D.H.
      ,
      • Teckman J.H.
      • An J.K.
      • Loethen S.
      • Perlmutter D.H.
      ,
      • Teckman J.H.
      • An J.K.
      • Blomenkamp K.
      • Schmidt B.
      • Perlmutter D.
      ,
      • Perlmutter D.H.
      ). Moreover, ER stress in yeast and mammals leads to induction of autophagy (
      • Bernales S.
      • McDonald K.L.
      • Walter P.
      ,
      • Yorimitsu T.
      • Nair U.
      • Yang Z.
      • Klionsky D.J.
      ).
      Because the majority of ERAD substrates are decorated by N-linked glycans, we postulated that attenuation in the degradation of certain ERAD substrates observed in the absence of PNG1 may lead to the recruitment of autophagic components to the ER lesion. To unravel a potential function of autophagy in the removal of glycosylated ER-misfolded proteins, we searched for a known ERAD substrate whose proteasomal clearance is attenuated in the Δpng1 strain in an autophagy-dependent manner. We found that in the absence of PNG1, the degradation of 6Myc-Hmg2p, an ERAD-M substrate, is attenuated. This inhibition is inferred from the formation of a stable complex between the ERAD substrate and components of the autophagic machinery. Finally, we demonstrate that upon deletion of the unstructured 23 C-terminal residues of 6Myc-Hmg2p, a portion of this substrate travels to the vacuole in an autophagy-dependent manner. Taken together, these results provide evidence for a new role for the autophagic machinery in ER quality control of transmembrane proteins.

      DISCUSSION

      Although the role of the proteasome in the degradation of ER-misfolded proteins has been established, the involvement of the lysosomes/vacuole, the other major degradative cellular pathway, has remained unclear. Here we present evidence for an alternative route for clearance of an ERAD substrate by the vacuole. By investigating the removal of 6Myc-Hmg2p, one of the most studied ERAD-M substrates, we identified an interaction between the misfolded ubiquitinated Hmg2p and elements of the CVT machinery on the ER membrane (Figs. 3A and 4B). This interaction is destabilized by the action of Png1, the cytosolic N-glycanase, previously implicated in ER-associated degradation (
      • Kim I.
      • Ahn J.
      • Liu C.
      • Tanabe K.
      • Apodaca J.
      • Suzuki T.
      • Rao H.
      ,
      • Hosomi A.
      • Tanabe K.
      • Hirayama H.
      • Kim I.
      • Rao H.
      • Suzuki T.
      ,
      • Suzuki T.
      • Park H.
      • Hollingsworth N.M.
      • Sternglanz R.
      • Lennarz W.J.
      ). In the absence of PNG1, a stable complex is formed, which in turn leads to a slowdown in proteasomal degradation of 6Myc-Hmg2p (Fig. 1B). Concomitant removal of PNG1 and one of several key autophagic factors resumes fast proteasomal degradation of 6Myc-Hmg2p (Figs. 3, B and D, and 4A). We also demonstrate that the regulatory role of Png1 is mediated by the C terminus of Hmg2p. Truncation of the last 23 residues of the 6Myc-Hmg2p as well as the addition of a FLAG tag at its C terminus decoupled Png1 activity and proteasomal degradation of 6Myc-Hmg2p (Figs. 6B and 8A). This allows delivery of a portion of the truncated ERAD-M substrate 6Myc-Hmg2p-1–1022 to vacuolar degradation in an ATG8- and ATG19-dependent manner (Fig. 7). Based on these findings, we postulate the existence of a new route responsible for the delivery of ERAD substrates to the vacuole acting in parallel to the proteasome.
      Figure thumbnail gr8
      FIGURE 8A model for the involvement of Png1 and the autophagic machinery in regulating the degradation of 6Myc-Hmg2p. In the presence of an active Png1, 6Myc-Hmg2p is removed from the ER membrane to the cytosol, where it is rapidly degraded by the proteasome. This process entails retrotranslocation, ubiquitination, and extraction from the ER membrane followed by proteasomal degradation. In the absence of PNG1, autophagy- and CVT-specific components attenuate the degradation process, leading to the accumulation of ubiquitinated 6Myc-Hmg2p on the ER membrane.
      In the present study, we identified for the first time a physical interaction between an ERAD-M target and Atg8, a key autophagic factor (
      • Ichimura Y.
      • Kirisako T.
      • Takao T.
      • Satomi Y.
      • Shimonishi Y.
      • Ishihara N.
      • Mizushima N.
      • Tanida I.
      • Kominami E.
      • Ohsumi M.
      • Noda T.
      • Ohsumi Y.
      ). Atg8 has been implicated previously in selective recruitment of Ape1 and mitochondria into autophagosomes by interacting with Atg19 and Atg32, respectively (
      • Okamoto K.
      • Kondo-Okamoto N.
      • Ohsumi Y.
      ,
      • Kanki T.
      • Wang K.
      • Cao Y.
      • Baba M.
      • Klionsky D.J.
      ,
      • Scott S.V.
      • Guan J.
      • Hutchins M.U.
      • Kim J.
      • Klionsky D.J.
      ). Notably, we also found that 6Myc-Hmg2p co-immunoprecipitated with the precursor form of Ape1 (Fig. 4B), suggesting that these proteins together with Atg8 and potentially other factors form a complex on the ER membrane. Functional evidence for this interaction is also provided. Accordingly, the removal of either ATG8 or ATG19 alleviated the attenuated proteasomal degradation in Δpng1 (Figs. 3A and 4A). Likewise, Atg3 and Atg7, factors mediating Atg8 conjugation to phosphatidylethanolamine (
      • Ichimura Y.
      • Kirisako T.
      • Takao T.
      • Satomi Y.
      • Shimonishi Y.
      • Ishihara N.
      • Mizushima N.
      • Tanida I.
      • Kominami E.
      • Ohsumi M.
      • Noda T.
      • Ohsumi Y.
      ), are also involved in this process, indicating that lipidation of Atg8 may also be required. Moreover, delivery of 6Myc-Hmg2p lacking the last 23 amino acids to the vacuole was found to be fully dependent on ATG8 and ATG19 (Fig. 7B). These findings are consistent with a recent report implicating the CVT pathway in degradation of a mutated misfolded form of Pma1 (
      • Mazón M.J.
      • Eraso P.
      • Portillo F.
      ).
      One of the consequences of elevated 6Myc-Hmg2p levels in the ER membrane was reflected by prevention of vacuolar transport of GFP-Atg8 and CVT cargo (Fig. 5, A, B, and D). Most likely, under normal growth conditions, crucial components of these pathways present in limiting amounts, such as Atg8 and Atg19, are recruited to the ER membrane lesion, rendering them unavailable for the constitutive CVT. Consistently, expression of 6Myc-Hmg2p in wild-type cells led to a 2.6-fold increase in the transcription level of ATG8 (supplemental Fig. S5), perhaps to partially compensate for the sequestration of Atg8. Blockage of starvation-induced transport of GFP-Atg8 supports the notion that in the absence of PNG1, a stable complex between 6Myc-Hmg2p and Atg8 is formed. Our findings indicate that both expression of 6Myc-Hmg2p and the absence of PNG1 are necessary for the attenuation of vacuolar delivery of GFP-Atg8 upon starvation (Fig. 5, A and B). This was unexpected because nitrogen depletion induces strong up-regulation of the ATG8 transcript (
      • Kirisako T.
      • Baba M.
      • Ishihara N.
      • Miyazawa K.
      • Ohsumi M.
      • Yoshimori T.
      • Noda T.
      • Ohsumi Y.
      ) (supplemental Fig. S5) that should compensate for the fraction of GFP-Atg8 recruited to 6Myc-Hmg2p. Together, these findings suggest that the GFP-Atg8 sequestrated by the ERAD-M substrate under normal growth conditions is tightly bound. Thus, upon starvation, the elevated endogenous Atg8 expression did not chase off GFP-Atg8 from the ER membrane, thereby not reaching the vacuole. In support, when GFP-Atg8 was expressed under the endogenous ATG8 promoter, its levels were elevated upon starvation, and GFP was detected in the vacuole in Δpng1 yeast expressing 6Myc-Hmg2p (supplemental Fig. S7). Based on these findings, we conclude that in the absence of PNG1, the complex between Atg8 and 6Myc-Hmg2p is stabilized.
      Here we provide evidence for a new role of the CVT machinery in surveying the ER membrane for potential aberration. Our data imply that this system acts differently depending on the character of the misfolded protein found on the ER membrane. In one scenario, where 6Myc-Hmg2p is expressed in cells lacking PNG1, the CVT machinery may fulfill a protective function, sequestering the lesion from the bulk cytosol. Indeed, Bernales et al. (
      • Bernales S.
      • McDonald K.L.
      • Walter P.
      ) reported that an elevation in cellular autophagy in response to ER stress might also lead to the appearance of membrane expansions and autophagosome-like structures. Under these conditions, the vesicles are thought to play a protective role in response to ER stress induced by the addition of DTT or tunicamycin rather than delivering the content for vacuolar degradation. Truncation of the 6Myc-Hmg2p C-terminal 23-amino acid stretch constituted a different scenario whereby a significant portion of this ERAD substrate is delivered to vacuolar degradation in a CVT-dependent manner. Either way, our data imply that recruitment and commitment of the autophagic system to a membrane lesion in the ER is relatively slow compared with the proteasomal degradation associated with ER dislocation. Upon slowdown in the extraction of the defective protein, the association with Atg8 and the CVT complex is stabilized, thus providing an autophagy-dependent checkpoint for the extraction and degradation process of 6Myc-Hmg2p (Fig. 8).
      ER chaperones were previously demonstrated to maintain ERAD substrates in a retrotranslocation-competent state (
      • Brodsky J.L.
      • Werner E.D.
      • Dubas M.E.
      • Goeckeler J.L.
      • Kruse K.B.
      • McCracken A.A.
      ,
      • Casagrande R.
      • Stern P.
      • Diehn M.
      • Shamu C.
      • Osario M.
      • Zúñiga M.
      • Brown P.O.
      • Ploegh H.
      ). Moreover, these studies concluded that the ERAD machinery is unable to process aggregated targets. It appears that a selective set of chaperones is required for retrotranslocation of different ER aberrant proteins (
      • Nishikawa S.I.
      • Fewell S.W.
      • Kato Y.
      • Brodsky J.L.
      • Endo T.
      ). Interestingly, mammalian PNGase was shown to associate with the ERAD machinery promoting retrotranslocation (
      • Zhao G.
      • Zhou X.
      • Wang L.
      • Li G.
      • Schindelin H.
      • Lennarz W.J.
      ,
      • Katiyar S.
      • Joshi S.
      • Lennarz W.J.
      ). Accordingly, Png1 promotes efficient extraction of 6Myc-Hmg2p, thus acting as chaperone in this system. In fact, we show here that upon deletion of PNG1, the ERAD-M substrate accumulates on the membrane decorated with polyubiquitin, in Triton X-100-resistant aggregates (Fig. 2B). Deletion of the 6Myc-Hmg2p C terminus (6Myc-Hmg2p-1–1022-RFP) renders the protein insensitive to the presence of Png1, leading to its partial delivery to the vacuole (Fig. 7). We argue that the fraction of 6Myc-Hmg2p-1–1022-RFP that was delivered to the vacuole may form aggregates on the ER inaccessible to the ERAD machinery.
      In the present study, we provide evidence of a new role for an autophagy-related mechanism in the selective removal of an ERAD-M substrate. Support for the selectivity of the process emerges from the fact that Atg19, a protein implicated in the selective delivery of Ape1 from cytosol to vacuole, was found to be essential for the delivery of 6Myc-Hmg2p-1–1022-RFP to the vacuole. Moreover, we show that Ape1, in its premature form, associates with 6Myc-Hmg2p on the ER membrane to form a complex that also contains Atg8. Based on these findings and the functional analysis of the effect of the different ATG null mutants on 6Myc-Hmg2p degradation, we hypothesize that factors of the CVT machinery constantly survey the ER membrane for possible lesions caused by the accumulation of transmembrane proteins. Once such a lesion is recognized, possibly by interaction of Atg8 and Ape1 with ubiquitinated ERAD-M substrates, a complex is formed. Under normal conditions, Png1, acting as a chaperone, is responsible for the dissociation of this complex, thus allowing the retrotranslocation of the ERAD-M substrate and its delivery for proteasomal degradation. In the absence of Png1, the complex between 6Myc-Hmg2p and factors of the CVT machinery is stabilized, leading to a slowdown in the degradation of the ERAD-M substrate, possibly playing a protective role of the ER lesions.
      Autophagy was originally thought to act non-selectively; however, a growing body of evidence provides examples of molecular mechanisms for cargo specificity. In yeast, numerous selective autophagy pathways were recently described. For instance, Atg19 acts as a specific receptor for the delivery of Ape1 and Ams1 from the cytosol to the vacuole via the CVT pathway (
      • Scott S.V.
      • Guan J.
      • Hutchins M.U.
      • Kim J.
      • Klionsky D.J.
      ,
      • Shintani T.
      • Huang W.P.
      • Stromhaug P.E.
      • Klionsky D.J.
      ). Peroxisome recruitment into autophagosomes depends on Pex3 and Pex14, two peroxisomal proteins essential for targeting peroxisomes for autophagy (
      • Farré J.C.
      • Manjithaya R.
      • Mathewson R.D.
      • Subramani S.
      ). The expansion of the ER triggered by the unfolded protein response is remodeled to its homeostatic volume by selective autophagy (
      • Bernales S.
      • McDonald K.L.
      • Walter P.
      ). Selective degradation of the 60 S ribosomal subunit by the autophagic pathway is regulated by the ubiquitin protease Ubp3 and its activator Bre5 (
      • Kraft C.
      • Deplazes A.
      • Sohrmann M.
      • Peter M.
      ). Finally, Atg32, a mitochondrial peripheral membrane protein, was recently reported to target autophagosomes to mitochondria by interacting with Atg8 and Atg11 (
      • Okamoto K.
      • Kondo-Okamoto N.
      • Ohsumi Y.
      ,
      • Kanki T.
      • Wang K.
      • Cao Y.
      • Baba M.
      • Klionsky D.J.
      ). Based on our findings, we propose that the autophagic machinery selectively recognizes the ERAD M substrate 6Myc-Hmg2p, thus implicating selective autophagy during ERAD. Future experiments are required to determine whether this system represents part of a more general function of the CVT machinery in quality control of the ER membrane.

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