Membrane Protein Biogenesis: Regulated Complexity at the Endoplasmic Reticulum

The enormous diversity of proteins that transit the secretory pathway demands a mechanistic complexity in biogenesis that has yet to be fathomed. The current understanding of the molecular components that direct the translocation of a limited subset of simple secretory proteins has revealed a remarkably (and probably deceptively) simple picture. As the lessons learned from these studies are being extended to slightly more complex substrates, it is becoming more and more obvious that our understanding is neither complete nor clear. Many substrates, especially membrane proteins, appear to require the translocation machinery to make “decisions” specific to a particular situation or substrate: Which domains of a protein are TM segments? Which TM segments should be held in the translocation channel for purposes of folding or association with distal domains, and which should be integrated immediately into the bilayer? What should the orientation of various TM segments be? With each of these decisions comes the opportunity for regulation.Although fundamental advances toward answering such questions will undoubtedly require the development of new ideas as well as techniques, a handful of initial studies on complex substrates may suggest the functional regulation of protein biogenesis. As with transcriptional and translational control, the cell may use translocational control as an additional means of generating diversity of gene expression. Indeed, several membrane proteins have been observed to be expressed in multiple topological forms, with the diversity apparently being generated at the time of translocation at the ER membrane (seeLevy 1996xMembrane proteins which exhibit multiple topological orientations. Levy, D. Essay Biochem. 1996; 31: 49–60PubMedSee all ReferencesLevy 1996, for a review). For example, the protein ductin not only has two orientations (Finbow et al. 1993xDisposition and orientation of ductin (DCCD-reactive vacuolar H(+)-ATPase subunit) in mammalian membrane complexes. Finbow, M.E., John, S., Kam, E., Apps, D.K., and Pitts, J.D. Exp. Cell Res. 1993; 207: 261–270Crossref | PubMedSee all ReferencesFinbow et al. 1993), but each orientation appears to serve different functions. One of the topological forms is found as a subunit of the vacuolar H+-ATPase, while the other form is a component of a connexon channel found in gap junctions. That this diversity originates at the translocation site in the ER was demonstrated by showing that ductin translated and translocated in a cell-free system results in the synthesis in both orientations (Dunlop et al. 1995xMembrane insertion and assembly of ductin (a polytopic channel with dual orientations) . Dunlop, J., Jones, P.C., and Finbow, M.E. EMBO J. 1995; 14: 3609–3616PubMedSee all ReferencesDunlop et al. 1995).Similarly, the P-glycoprotein product of the multidrug resistance gene (MDR1) found in various cancer cells is a membrane protein with at least two topological forms (40xEvidence for an alternate model of human P-glycoprotein structure and biogenesis. Skach, W.R., Calayag, M.C., and Lingappa, V.R. J. Biol. Chem. 1993; 268: 6903–6908PubMedSee all References, 49xMembrane topology of the N-terminal half of the hamster P-glycoprotein molecule. Zhang, J.T., Duthie, M., and Ling, V. J. Biol. Chem. 1993; 268: 15101–15110PubMedSee all References). Although predicted to span the membrane 12 times, several of its TM segments apparently can exist in multiple orientations or locations, perhaps regulated by factors in the cytosol (Zhang and Ling 1995xInvolvement of cytoplasmic factors regulating the membrane orientation of P-glycoprotein sequences. Zhang, J.T. and Ling, V. Biochem. 1995; 34: 9159–9165Crossref | PubMedSee all ReferencesZhang and Ling 1995). This type of structural variability appears to be qualitatively different than that observed in ductin, where the entire protein is reversed in orientation with respect to the membrane. However, similar to ductin, MDR1 has been proposed to serve multiple functions in the cell (Pastan and Gottesman 1991xMultidrug resistance. Pastan, I. and Gottesman, M.M. Annu. Rev. Med. 1991; 42: 277–286Crossref | PubMedSee all ReferencesPastan and Gottesman 1991). Whether the different topological forms are recruited to different regions of the cell for specialized functions, as appears to be the case for ductin, remains to be determined.Finally, some proteins may contain potential TM segments that are not used under all circumstances. For example, the prion protein (PrP), a brain glycoprotein involved in various neurodegenerative diseases (Prusiner 1996xMolecular biology and pathogenesis of prion diseases. Prusiner, S.B. Trends Biochem. Sci. 1996; 21: 482–487Abstract | Full Text PDF | PubMed | Scopus (218)See all ReferencesPrusiner 1996), contains a hydrophobic domain initially predicted to serve as a TM segment (Bazan et al. 1987xPredicted secondary structure and membrane topology of the scrapie prion protein. Bazan, J.F., Fletterick, R.J., McKinley, M.P., and Prusiner, S.B. Prot. Eng. 1987; 1: 125–135Crossref | PubMed | Scopus (43)See all ReferencesBazan et al. 1987). Despite this hydrophobic segment, PrP does not appear to normally span the membrane in vivo, but rather is translocated across the ER membrane, C-terminally glycolipididated, and trafficked to the cell surface (Stahl et al. 1987xScrapie prion protein contains a phosphatidylinositol glycolipid. Stahl, N., Borchelt, D.R., Hsiao, K., and Prusiner, S.B. Cell. 1987; 51: 229–240Abstract | Full Text PDF | PubMed | Scopus (686)See all ReferencesStahl et al. 1987). By contrast, studies in cell-free systems have shown that not only can PrP span the membrane at its putative TM segment, but under some conditions, nearly all of it is found as a transmembrane protein (Hay et al. 1987xBiogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein. Hay, B., Barry, R.A., Lieberburg, I., Prusiner, S.B., and Lingappa, V.R. Mol. Cell. Biol. 1987; 7: 914–920PubMedSee all ReferencesHay et al. 1987). The generation of this topological form is dependent on both hydrophobic and hydrophilic sequences in the PrP molecule (Yost et al. 1990xNon-hydrophobic extracytoplasmic determinant of stop transfer in the prion protein. Yost, C.S., Lopez, C.D., Prusiner, S.B., Myers, R.M., and Lingappa, V.R. Nature. 1990; 343: 669–672Crossref | PubMedSee all ReferencesYost et al. 1990) and appears to be regulated by cytosolic factors (Lopez et al. 1990xUnusual topogenic sequence directs prion protein biogenesis. Lopez, C.D., Yost, C.S., Prusiner, S.B., Myers, R.M., and Lingappa, V.R. Science. 1990; 248: 226–229Crossref | PubMedSee all ReferencesLopez et al. 1990). However, just as the normal role of the PrP molecule remains enigmatic, so does the topological regulation of this unusual protein. It will be interesting to see whether the topology of PrP is regulated in vivo by trans-acting cellular factors, and whether dysregulation of these events at the ER plays a role in any of the wide variety of diseases attributed to PrP. If so, it seems likely that a transmembrane form, not being observed in normal brain, is involved in events related to prion disease that are carried out, in part, by as yet unidentified components of the translocon.The identification and functional reconstitution of the core components of the translocon, using simple substrates, have now set the stage for exploring the functional complexity and structural diversity of accessory translocon components in biogenesis of more complex secretory and membrane proteins. The initial studies on membrane proteins have already revealed unappreciated subtleties of the translocation process, pointing to a new site of regulation in the cell. Future work using the tools obtained from past work on model proteins, used to study the biogenesis of currently enigmatic substrates, will surely elucidate new functions for old machinery and new machinery involved in currently mysterious functions.