Molecular Cell

Previews ERQC Autophagy: Yet Another Way to Die Alexander Buchberger1,* 1Department

of Biochemistry, Biocenter, University of Wu¨rzburg, Am Hubland, 97074 Wu¨rzburg, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.03.037

A novel autophagy pathway eliminates nonnative polytopic membrane proteins from the endoplasmic reticulum that evade degradation by the ubiquitin proteasome system. Productive folding of newly translated proteins in the crowded intracellular environment is a highly vulnerable process endangered by misfolding and aggregation (Dobson, 2003). Because malfunctioning and aggregated proteins pose a serious threat to cell survival, all organisms possess potent protein quality control systems to cope with protein damage. These systems comprise molecular chaperones suppressing aggregation and assisting productive folding, proteolytic systems eliminating irreparably damaged proteins, and gene expression programs for adaptive responses to protein folding stress (Buchberger et al., 2010). In eukaryotes, folding of proteins in the endoplasmic reticulum (ER) is particularly complicated due to concomitant glycosylation, disulfide bond formation, and membrane insertion, making the ER a focal point of protein quality control, termed ERQC (Buchberger et al., 2010; Araki and Nagata, 2011). Importantly, impaired or overly tight ERQC gives rise to human diseases like cystic fibrosis, liver failure, and retinitis pigmentosa (Hebert and Molinari, 2007; Perlmutter, 2011). While the ER contains an arsenal of molecular chaperones, it lacks proteases for the elimination of irreparable proteins, necessitating their transport to two proteolytic systems outside the ER, the 26S proteasome and the lysosome (Figure 1). In the ER-associated protein degradation (ERAD) pathway, un-/misfolded luminal and membrane proteins are targeted to ER membraneembedded E3 ubiquitin ligase complexes for covalent modification with ubiquitin chains. For the extensively studied polytopic membrane protein cystic fibrosis transmembrane conductance regulator (CFTR), this process involves the ERADspecific E3 RMA1, Derlin-1, cytosolic Hsp70 chaperone, and its single membrane-spanning Hsp40 cofactor DnaJB12

(Young, 2014). Ubiquitylated proteins are then dislocated from the ER membrane by the adenosine triphosphatase (ATPase) p97 and delivered to the 26S proteasome for degradation (Figure 1, route 1). By contrast, insoluble protein aggregates like that formed by mutant a1-antitrypsin Z (ATZ) cannot be dislocated from the ER and are eliminated by macroautophagy (referred to as autophagy hereafter) instead (Perlmutter, 2011). This involves their

incorporation into double-membranebounded structures termed autophagosomes, which subsequently fuse with the lysosome to deliver their contents for degradation by lysosomal hydrolases (Figure 1, route 2) (Mizushima et al., 2011). Intriguingly, in this issue of Molecular Cell, an additional ERQC-autophagy pathway for the degradation of nonaggregated, but ERAD-resistant, membrane proteins is revealed (Houck et al., 2014).

Figure 1. Degradation Pathways for Membrane Proteins at the Endoplasmic Reticulum Polytopic membrane proteins (green) that fail to fold into their native conformation are eliminated from the ER by ERAD (red) or autophagy (blue). Un-/misfolded conformers (e.g., GnRHR-S168K, rhodopsin-P23H, and CFTR) are targeted to ERAD. They are recognized by the Hsp40 chaperone DNAJB12 (J) and cytosolic Hsp70 (70) and delivered to a complex containing Derlin-1 (Der) and the E3 ubiquitin ligase RMA1. After ubiquitylation, they are dislocated by the ATPase p97 and degraded by the 26S proteasome (route 1). Under conditions of limiting proteasomal capacity, dislocated substrates can aggregate in the cytosol and accumulate in aggresomes, which are delivered to the lysosome for degradation by selective autophagy (route 1a). ERAD-resistant protein aggregates (e.g., ATZ and Dysferlin) are engulfed by autophagosomes and degraded by lysosomal hydrolases upon fusion of the autophagosome with the lysosome (route 2). Nonaggregated ERAD-resistant conformers like GnRHR-E90K are recognized by Hsp70-DNAJB12, but not ubiquitylated or dislocated. Instead, they form a complex with Beclin-1 and hVps34 and are sorted into autophagosomes (route 3). For a comprehensive account of folding and degradation of luminal ER proteins (omitted here for clarity), see Buchberger et al. (2010) and Araki and Nagata (2011).

Molecular Cell 54, April 10, 2014 ª2014 Elsevier Inc. 3

Molecular Cell

Previews Houck et al. (2014) utilized wild-type and mutant forms of human gonadotropin-releasing hormone receptor type I (GnRHR) as model substrates. GnRHR is a member of the large family of G protein-coupled receptors (GPCRs), which uniformly possess seven transmembrane helices and are subject to tight quality control in the ER (Conn and Ulloa-Aguirre, 2011). Even for wild-type GnRHR, there is a delicate balance between plasma membrane delivery of the native, functional receptor and ER retention and degradation of nonnative conformers. A number of missense mutations tip the balance toward complete ER retention/degradation of GnRHR and are associated with the sexual maturation disorder hypogonadotropic hypogonadism (Online Mendelian Inheritance in Man [OMIM] 146110). Among those mutations, the S168R exchange results in global misfolding of GnRHR, whereas the E90K mutant receptor appears to represent a late, nearnative folding intermediate (Conn and Ulloa-Aguirre, 2011). Houck et al. (2014) found that wild-type and S168R mutant GnRHR behaved as prototypical ERAD substrates (Figure 1, route 1): they accumulated upon addition of proteasome, but not autophagy, inhibitors and were degraded more rapidly upon overexpression of the E3 ligase RMA1. Surprisingly, the E90K mutant receptor behaved fundamentally differently, as it was insensitive to RMA1 overexpression and stabilized by autophagy inhibitors, indicating that it is eliminated by autophagy rather than ERAD. Indeed, E90K localized to punctate structures in the cytosol that bear all hallmarks of autophagosomes. Importantly, the authors provide compelling evidence that the autophagic E90K degradation is distinct from the previously described autophagy pathways for ATZ (Figure 1, route 2) and for large cytosolic aggregates termed aggresomes (Figure 1, route 1a), respectively. They show that E90K, unlike ATZ, does not form detergent-insoluble aggregates, and that the E90K-positive punctate structures colocalize neither with autophagosomes containing ATZ aggregates nor with aggresomes. Furthermore, unlike the selective autophagy of aggresomes, autophagic degradation of E90K does not depend on substrate ubiquitylation and the autophagosome cargo receptors p62

and NBR1. Instead, E90K associates with DnaJB12, Hsp70, Beclin-1, and hVps34 (Figure 1, route 3). The latter two proteins are subunits of a phosphatidylinositol 3-kinase (PI3K) complex that is recruited to the ER membrane to produce phosphatidylinositol 3-phosphates as an essential first step in autophagosome formation (Mizushima et al., 2011). The association of E90K with Beclin-1 suggests that E90K is incorporated into nascent autophagosomes and degraded via bulk autophagy. Finally, Houck et al. (2014) explored which properties target E90K to the new degradation pathway. Blocking autophagosome formation with a PI3K inhibitor resulted in the accumulation of E90K at the ER membrane, but not in its degradation via ERAD, while destabilization of E90K converted it into an ERAD substrate. Contrariwise, blocking ERAD by inactivation of p97 redirected wild-type and S168K mutant GnRHR to the ERQC-autophagy pathway (Figure 1, dashed double arrow). Thus, ERQC autophagy eliminates substrates that adopt an ERAD-resistant conformation or are otherwise resistant to ERAD. The study by Houck et al. (2014) adds another facet to the fascinating strategies of ERQC by showing that nonnative, ERAD-resistant but nonaggregated ER membrane proteins can be eliminated by autophagy. This exciting new finding immediately raises important questions. First, what is the molecular basis underlying substrate selection for the new ERQCautophagy pathway? The authors favor the model that the near-native conformation of E90K itself is the critical determinant. This would require a specificity factor capable of distinguishing globally misfolded and native from near-native conformers of GnRHR and targeting only the latter. While the existence of such a factor cannot be excluded, the ERQC factors found so far to associate with E90K recognize a broad range of nonnative conformers. Moreover, a near-native conformation is unlikely to be strictly required for degradation by ERQC autophagy because the constitutively misfolded mutant S168K was redirected to ERQC autophagy upon blockage of ERAD. However, impaired dislocation may be an alternative determinant for sorting to ERQC autophagy. Failure of the ERAD system to recognize or ubiquitylate nonnative pro-

4 Molecular Cell 54, April 10, 2014 ª2014 Elsevier Inc.

teins or to dislocate ubiquitylated proteins from the ER prevents their proteasomal degradation and provokes their accumulation at the ER membrane. In one possible scenario, the Hsp70-DnaJB12 chaperone complex would recognize E90K as nonnative substrate, but RMA1 would fail to efficiently ubiquitylate it, leading to the accumulation of E90K-Hsp70-DnaJB12 complexes that are subsequently sorted for ERQC autophagy by association with Beclin-1 and hVps34. Effectively, this would constitute a kinetic competition model of pathway selection relying on the relative rates of ubiquitylation and/or dislocation versus delivery to autophagosomes. Further open questions relate to the mechanism of cargo incorporation into ERQC autophagosomes. Is prolonged dwell time on Hsp70-DnaJB12 and interaction with Beclin-1 and hVps34 sufficient for a passive incorporation into nascent autophagosomes, or is active sorting involving additional factors required? Are aggregated proteins like ATZ delivered to autophagosomes via Beclin-1 and hVps34 as well, or do they use a distinct mechanism? Finally, it appears highly unlikely that Hsp70-DnaJB12-mediated ERQC autophagy has exclusively evolved for the degradation of late GnRHR folding intermediates in humans. Rather, the study by Houck et al. (2014) sets the stage for the identification of additional wild-type and mutant GPCRs and other difficultto-fold membrane proteins that are subject to ERQC autophagy. REFERENCES Araki, K., and Nagata, K. (2011). Cold Spring Harb. Perspect. Biol. 3, a007526. Buchberger, A., Bukau, B., and Sommer, T. (2010). Mol. Cell 40, 238–252. Conn, P.M., and Ulloa-Aguirre, A. (2011). Adv. Pharmacol. 62, 109–141. Dobson, C.M. (2003). Nature 426, 884–890. Hebert, D.N., and Molinari, M. (2007). Physiol. Rev. 87, 1377–1408. Houck, S.A., Ren, H.Y., Madden, V.J., Bonner, J.N., Conlin, M.P., Janovick, J.A., Conn, P.M., and Cyr, D.M. (2014). Mol. Cell 54, this issue, 166–179. Mizushima, N., Yoshimori, T., and Ohsumi, Y. (2011). Annu. Rev. Cell Dev. Biol. 27, 107–132. Perlmutter, D.H. (2011). Annu. Rev. Med. 62, 333–345. Young, J.C. (2014). Dis. Model. Mech. 7, 319–329.

ERQC autophagy: Yet another way to die.

A novel autophagy pathway eliminates nonnative polytopic membrane proteins from the endoplasmic reticulum that evade degradation by the ubiquitin prot...
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