Vishwanath R. Lingappa

Rapid regulation of steroidogenesis by mitochondrial protein import.

Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and imported into mitochondria. The imported proteins are directed to one of four submitochondrial compartments—the outer mitochondrial membrane, the inner mitochondrial membrane, the intramembraneous space, or the matrix—where the protein then functions. Here we show that the steroidogenic acute regulatory protein (StAR), a mitochondrial protein required for stress responses, reproduction, and sexual differentiation of male fetuses, exerts its activity transiently at the outer mitochondrial membrane rather than at its final resting place in the matrix. We also show that its residence time at this outer membrane and its activity are regulated by its speed of mitochondrial import. This may be the first example of a mitochondrial protein exerting its biological activity in a compartment other than that to which it is finally targeted. This system enables steroidogenic cells to initiate and terminate massive levels of steroidogenesis within a few minutes, permitting the rapid regulation of serum steroid hormone concentrations.

Transmissible and genetic prion diseases share a common pathway of neurodegeneration.

Prion diseases can be infectious, sporadic and genetic. The infectious forms of these diseases, including bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, are usually characterized by the accumulation in the brain of the transmissible pathogen, an abnormally folded isoform of the prion protein (PrP) termed PrPSc. However, certain inherited PrP mutations appear to cause neurodegeneration in the absence of PrPSc (refs 5,6,7,8), working instead by favoured synthesis of CtmPrP, a transmembrane form of PrP (ref. 9). The relationship between the neurodegeneration seen in transmissible prion diseases involving PrPSc and that associated with CtmPrP has remained unclear. Here we find that the effectiveness of accumulated PrPSc in causing neurodegenerative disease depends upon the predilection of host-encoded PrP to be made in the CtmPrP form. Furthermore, the time course of PrPSc accumulation in transmissible prion disease is followed closely by increased generation of CtmPrP. Thus, the accumulation of PrPSc appears to modulate in trans the events involved in generating or metabolising CtmPrP. Together, these data suggest that the events of CtmPrP-mediated neurodegeneration may represent a common step in the pathogenesis of genetic and infectious prion diseases.

Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]

Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino- or the carboxy-terminal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.

Regulation of Protein Topology by trans-Acting Factors at the Endoplasmic Reticulum

In mammalian cells, the Sec61 complex and translocating chain-associated membrane protein (TRAM) are necessary and sufficient to direct the biogenesis, in the appropriate topology, of all secretory and membrane proteins examined thus far. We demonstrate here that the proper translocation of the prion protein (PrP), a substrate that can be synthesized in more than one topologic form, requires additional factors. In the absence of these additional factors, PrP is synthesized exclusively in the transmembrane topology (termed the CtmPrP form) associated with the development of neurodegenerative disease. Thus, translocation accessory factors, acting on some but not other substrates, can function as molecular switches to redirect nascent proteins toward divergent topologic fates with different functional consequences.

A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems

We have combined molecular genetic and cell-free reconstitution approaches to study the mechanism of membrane assembly. The coding region for the carboxy-terminal transmembrane sequence of membrane IgM heavy chain has been inserted between the coding regions for lactamase and globin domains of a fusion protein previously shown to be completely translocated across microsomal membranes in a cell-free transcription-linked translation system. The resulting fusion protein behaves as an integral transmembrane protein of predicted asymmetry: all of the membrane integrated copies display lactamase within the lumen and globin on the cytoplasmic face of the vesicles. In another construction, this transmembrane coding region replaces that of the signal sequence. The resulting fusion protein is not translocated across membranes. These data provide strong evidence that there are stop transfer sequences whose ability to arrest chain translocation and achieve asymmetric transmembrane orientation is independent of the size of the subsequent carboxy-terminal domain to be localized in the cytosol; and that signal and stop transfer sequences are functionally distinct.

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.

TRAM Regulates the Exposure of Nascent Secretory Proteins to the Cytosol during Translocation into the Endoplasmic Reticulum

Translocational pausing is a mechanism used by certain specialized secretory proteins whereby discrete domains of a nascent chain destined for the endoplasmic reticulum lumen are transiently exposed to the cytosol. Proteoliposomes reconstituted from total endoplasmic reticulum proteins properly assemble translocationally paused intermediates. The capacity of the translocon to correctly pause the nascent chain is dependent on a glycoprotein fraction whose active component is TRAM. In the absence of TRAM, the normally sealed ribosome-membrane junction still opens in response to a pause transfer sequence. However, nascent chain domains that are not exposed to the cytosol in the presence of TRAM are so exposed in its absence. Thus, TRAM regulates which domains of the nascent chain are visible to the cytosol during a translocational pause.

Sequence-Specific Alteration of the Ribosome–Membrane Junction Exposes Nascent Secretory Proteins to the Cytosol

Tight docking of the ribosome at the translocation channel ensures that nascent secretory proteins are shielded from the cytoplasm during transfer into the endoplasmic reticulum. Discrete pause transfer sequences mediate the transient stopping of translocation in certain proteins. Here we show that during a translocational pause, the junction between the ribosome and translocation channel is opened, exposing the nascent chain to the cytosol. While transient, this opening is sufficient to demonstrate macromolecular interactions between the translocating chain and molecules added to the cytosol, such as antibodies and site-specific proteases. Moreover, this opening is accompanied by alterations in the proteins that neighbor the nascent chain. These results demonstrate that specific sequences within a translocating nascent chain can elicit dramatic and reversible structural changes in the translocation machinery. Thus, the translocon is dynamic and can be regulated.

Regulation of protein biogenesis at the endoplasmic reticulum membrane

The biogenesis of most secretory and membrane proteins involves targeting the nascent protein to the endoplasmic reticulum (ER), translocation across or integration into the ER membrane and maturation into a functional product. The essential machinery that directs these events for model secretory and membrane proteins has been identified, shifting the focus of studies towards the molecular mechanisms by which these core components function. By contrast, regulatory mechanisms that allow certain proteins to serve multiple functions within a cell remain entirely unexplored. This article examines each stage of protein biogenesis as a potential site of regulation that could be exploited by the cell to effectively increase the diversity of functional gene expression.

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome–translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.