ER quality control: the cytoplasmic connection.

The notion that the ER membrane participates in the retrograde translocation of proteins is not new. Proteins that are unable to complete translocation have been observed to accumulate at the cytoplasmic side of the ER membrane as signal peptidase–cleaved intermediates (6xGarcia, P.D, Ou, J.H, Rutter, W.J, and Walter, P. J. Cell Biol. 1988; 106: 1093–1104Crossref | PubMedSee all References, 10xNguyen, T.H, Law, D.T, and Williams, D.B. Proc. Natl. Acad. Sci. USA. 1991; 88: 1565–1569Crossref | PubMedSee all References). Because the active site of signal peptidase is present at the lumenal side of the ER membrane, these results suggest that at least the early steps of translocation are reversible. In addition, toxins like ricin or Shiga, which enter the cell through endocytosis, travel in retrograde through the secretory pathway to the ER and must be translocated across the ER membrane to gain access to the cytoplasm (Sandvig and van Deurs 1994xSandvig, K and van Deurs, B. FEBS Lett. 1994; 346: 99–102Abstract | Full Text PDF | PubMed | Scopus (65)See all ReferencesSandvig and van Deurs 1994). The mechanism of this translocation is unknown but may involve reversal of the translocation machinery, or perhaps a distinct transport system.In principle, the same set of general rules that govern the translocation of proteins into the ER (and across other membranes such as mitochondrial and bacterial inner membranes) should apply to the process of dislocation. Thus any dislocation mechanism should satisfy at least the following four criteria.First, dislocation substrates should be unfolded or loosely folded. Misfolded proteins that are destined for dislocation from the ER are associated with ER chaperones like BiP and calnexin, which prevent the formation of aggregates on the cis side of the ER membrane and can be envisioned to effectively maintain the proteins in a “dislocation competent” state. In CMV-infected cells, the US2 or US11 gene products, which encode type I integral membrane proteins could provide such a chaperone function.Second, there should be a source of energy to drive dislocation. On the trans (i.e., cytoplasmic) side of the membrane, candidate ATPases that could power dislocation include cytoplasmic HSP70 and the 19S cap of the proteasome, which possesses intrinsic unfolding ATPase activity. Proteasomes are bound to the cytoplasmic face of the ER membrane, where they could degrade polypeptides codislocationally. The role of ER-bound proteasomes in dislocation and degradation of lumenal and membrane proteins is an important area for future investigation.Third, there must be a transmembrane pore capable of engaging dislocating polypeptides and delivering them to the cytoplasmic machinery. The SecY/E protein complex, which forms the transmembrane pathway through which exported substrates are translocated across the bacterial inner membrane, can in the absence of proton motive force or association with the peripheral ATPase component, SecA, facilitate the backwards translocation of substrates (Schiebel et al. 1991xSchiebel, E, Driessen, A.J, Hartl, F.U, and Wickner, W. Cell. 1991; 64: 927–939Abstract | Full Text PDF | PubMed | Scopus (277)See all ReferencesSchiebel et al. 1991). Can the eukaryotic homolog of SecY/E, Sec61p, also function as a bidirectional translocator? Wiertz et al. 1996bxWiertz, E.J, Tortorella, D, Bogyo, M, Yu, J, Mothes, W, Jones, T.R, Rapoport, T.A, and Ploegh, H.L. Nature. 1996; 384: 432–438Crossref | PubMed | Scopus (848)See all ReferencesWiertz et al. 1996b reported that in US2-transfected cells treated with lactacystin to block proteolysis, deglycosylated MHC class I HCs can be coimmunoprecipitated in a complex together with US2 and Sec61p, consistent with the hypothesis that the Sec61p complex may provide a dislocation pathway across the membrane. Additional studies will be needed, however, to confirm whether or not those complexes represent bona fide dislocation intermediates, and whether or not additional membrane components are required to form the transmembrane pathway.Fourth and least well understood, there must be a mechanism by which dislocation substrates are recognized and delivered to the dislocation apparatus. Most translocation systems require a signal on the substrate protein or on an associated protein that is recognized by a receptor associated with the translocation pore. What sort of signal sequence directs dislocation substrates to the pore? The simplest model would be if misfolded substrates are never fully released from the translocation apparatus. Cross-linking studies suggest that release of integral membrane proteins from the translocation pore proceeds in a stepwise fashion, involving sequential interactions with Sec61p and TRAM and perhaps other components (Do et al. 1996xDo, H, Falcone, D, Lin, J, Andrews, D.W, and Johnson, A.E. Cell. 1996; 85: 369–378Abstract | Full Text | Full Text PDF | PubMed | Scopus (130)See all ReferencesDo et al. 1996). Perhaps a signal such as multiubiquitination prevents a misfolded protein from being fully released from the translocation apparatus and directs it to the proteasome. Such a situation may well operate for proteins like CFTR that expose large domains to the cytoplasmic ubiquitination apparatus and are rapidly degraded without detectable lag. At the other extreme are secretory proteins like prepro alpha factor, CPY*, and α1-antitrypsin, which, when correctly processed, should be protected from the cytoplasm by the ER membrane. If these polypeptides are indeed fully translocated and dissociated from the translocation apparatus, there must be an additional signal to direct them to the dislocation and the ubiquitination machinery. The coordination between the presence of misfolded proteins in the ER and the activation of the cytoplasmic ubiquitin machinery could involve the IRE1 gene, which encodes a transmembrane kinase that is required for the “unfolded protein response” in which the presence of unfolded proteins within the ER initiates the transcription of stress response genes in the nucleus (Cox et al. 1993xCox, J.S, Shamu, C.E, and Walter, P. Cell. 1993; 73: 1197–1206Abstract | Full Text PDF | PubMed | Scopus (660)See all ReferencesCox et al. 1993). Alternatively, retention of proteins in the immediate proximity of the dislocation–translocation apparatus, possibly mediated by an interaction between molecular chaperones and the dislocation apparatus, could eliminate the need for such targeting.Finally, how does the quality control machinery discriminate between proteins that are in the process of folding and those that are misfolded and should be degraded? Molecular chaperones like BiP and Hsp70 undergo cycles of ATP-dependent release from substrates; rebinding thus represents a kinetic partition between folded and unfolded states. These chaperones bind to folding intermediates and to misfolded proteins alike, suggesting that their presence or absence from a polypeptide chain cannot be the sole determinant of its fate. Some additional signal, possibly ubiquitination, must exist to allow the quality control machinery to evaluate the progress of the chaperone interaction and to keep chaperones from cycling indefinitely on nonproductive substrates. Perhaps protein fate is ultimately determined by a three-way kinetic partitioning between folding, chaperone binding, and multiubiquitination.Eukaryotic cells have evolved an amazing variety of editing and quality control systems that monitor the fidelity of key molecular events such as transcription, replication, and translation—and protein folding. It is now recognized that aberrant protein folding is an important etiologic factor in the pathogenesis of diseases including cystic fibrosis, α1-antitrypsin deficiency, retinitis pigmentosa, Alzheimer's disease, and prion diseases. The finding of a role for the ubiquitin–proteasome pathway in the degradation of proteins that fail to fold productively in the endoplasmic reticulum now adds one more function to the rapidly growing list of cellular roles for this versatile system and, for the first time, provides a molecular basis for understanding the quality control systems that operate on proteins in the secretory pathway.