Polytopic membrane proteins constitute an important class of macromolecules, representing ~20–30% of the proteome (Wallin and von Heijne, 1998
). Because of their complexity they are prone to misfold, and indeed many proteins in this class traffic inefficiently beyond the ER (Brodsky, 2007
). To better understand the pathway by which these proteins are destroyed, each step during the ERAD of Ste6p* was reconstituted. Based on in vivo studies and our findings, a model for the ERAD of Ste6p* is shown in . While it was previously known that the cytoplasmic Hsp70, Ssa1p, is required for ERAD, we demonstrate that the chaperone facilitates substrate-E3 ligase interaction. After ubiquitination, Ste6p* is captured by Cdc48p and released into the cytosol. Next, Ufd2p enhances the extent of ubiquitination. Finally, the soluble, species is a substrate for proteasome-mediated processing. Our results suggest that polytopic membrane proteins are ubiquitinated at the ER membrane but may be released into the cytosol for degradation.
A proposed pathway for the ERAD of a polytopic membrane protein
One view of chaperone function is that they bind misfolded substrates and prevent aggregate formation, thereby promoting recognition by the ubiquitination machinery. However, recent studies suggest that cytoplasmic chaperones form a high-order network and escort substrates to folding or degradative pathways (Albanese et al., 2006
; McClellan et al., 2005
; Wang et al., 2006
). Indeed, we found that Ste6p* remains detergent extractable after Ssa1p inactivation, and Ste6p*’s interaction with Doa10p—and its ubiquitination and degradation competence—are resurrected upon Ssa1p reactivation. In the absence of functional Ssa1p, Ste6p* solubility may be maintained by other chaperones, such as Hsp90 (Hsp82p in yeast, Figure S9
). Therefore, a multi-chaperone assembly may prevent the aggregation of ERAD substrates, and Hsp70 is most likely a component of this complex (, but note that distinct members of the complex may bind in a mutually exclusive manner). We also suggest that Hsp70 promotes Ste6p* recognition by Doa10p. At this time, it is not clear if Doa10p similarly recognizes a nonnative polypeptide motif in ERAD substrates, as proposed for Hrd1p (Bays et al., 2001
), or instead detects a degradation signal (Gardner and Hampton, 1999
; Ravid et al., 2006
Our inability to complement the Ste6p* ubiquitination defect in Hsp70 mutant microsomes upon the addition of WT cytosol was initially surprising (), especially since the ubiquitination and degradation of Ste6p* could be restored by Hsp70 reactivation in vivo. Moreover, repeated attempts to rescue the ssa1-45 mutant phenotype upon the addition of purified proteins were unsuccessful. We propose three models to explain these results. First, Ste6p* that resides in the Hsp70 mutant microsomes may be associated with an inert chaperone complex, and thus externally supplied Hsp70 would be sterically restricted for substrate access. Second, Hsp70 may facilitate Ste6p*-Doa10p interaction and ubiquitination by activating membrane-integrated factors. Therefore, WT cytosol would be unable to rescue the mutant phenotype. Third, Ste6p* in ssa1-45 mutant microsomes may lack a critical component which could not be supplied from cytosol or purified chaperones. Nevertheless, we cannot completely exclude the possibility that Hsp70 modulates Doa10p by subtly altering its conformation. However, we note that the defect in Doa10p-Ste6p* association in ssa1-45 microsomes was observed using two different crosslinkers, and that Doa10p solubility is unchanged when microsomes are prepared from either WT or ssa1-45 mutant yeast (data not shown).
It is not completely clear how the ER quality control machinery surveys integrating membrane proteins to identify those that are inappropriately assembled. Because Ste6p* ERAD was recovered upon Ssa1p reactivation and after translation arrest, we suggest that Doa10p recognizes Ste6p* post-translationally. This view is consistent with the fact that Ste6p* lacks the C-terminal 42 amino acids found in the full length, WT protein; thus, the conformations of translation and translocation intermediates of Ste6p* and WT Ste6p are identical, and the nature of the Ste6p* misfolding defect should only be evident post-translationally.
Several lines of evidence indicate that Doa10p is the ligase for membrane substrates with misfolded cytoplasmic domains, such as Ste6p* (Carvalho et al., 2006
; Vashist and Ng, 2004
). However, degradation and ubiquitination is incompletely inhibited when Doa10p is absent, which suggests that Hrd1p contributes to the ubiquitination of this class of substrates (Huyer et al., 2004
; this study). One means to explain this observation is that cytoplasmic domain misfolding might influence the assembly of the intermembrane domain, which is recognized by the Hrd1p complex. Alternatively, the Hrd1p complex may recognize a cytoplasmic misfolded region. Substrates for Hrd1p are not restricted to lumenal and membrane proteins (Arteaga et al., 2006
). This notion is also supported by the fact that the degradation of a general amino acid permease requires both Hrd1p and Doa10p (Kota et al., 2007
). Overall, depending on the topology and the location of the misfolded lesion, complex polytopic membrane proteins could be sorted to redundant pathways, which converge at the proteasome.
A long-standing question is whether polytopic ERAD substrates are degraded by the proteasome in situ at the membrane or whether they are extracted prior to proteolysis. Because solubilized, polyubiquitinated Ste6p* was precipitated with anti-HA antibody, which recognizes a lumenally disposed epitope in Ste6p*HA, our results indicate that the transmembrane domain of Ste6p* became solvent-exposed. More generally, our data provide the first in vitro evidence that aggresomes may form from the retro-translocation of a polytopic protein in the ER membrane.
It is unclear whether polytopic membrane proteins are intact or clipped prior to or during extraction. We note that the cytoplasmic polyubiquitinated species begin at and are higher than the molecular weight of the native species, suggesting strongly that unclipped proteins are released into the cytosol. If so, how is the substrate pulled from the ER? And, how is the solubility of the membrane domain maintained? The degradation of Ste6p* does not require proposed retro-translocation channels (Huyer et al., 2004
; Kreft et al., 2006
). Thus, Ste6p* could either be retro-translocated through an ill-defined channel or from the membrane. It should be noted that fusion of green fluorescent protein (GFP) to Ste6p* juxtaposed to the HA epitope tag in the ER lumen does not impair degradation (Huyer et al., 2004
), suggesting that the GFP moiety can be retro-translocated or extracted. Alternatively, the reconfiguration of membrane lipids could support extraction/release, and attached lipids may maintain the solubility of transmembrane domains (Ploegh, 2007
). Candidates for proteinaceous factors that could maintain the solubility of transmembrane domains include cytoplasmic chaperones, Cdc48p, proteasome-associated factors such as Rad23p/Dsk2p (Richly et al., 2005
), and the 19S particle. The in vitro system reported in this study provides a means to address these questions.