Determination of the Transmembrane Topology of Yeast Sec61p, an Essential Component of the Endoplasmic Reticulum Translocation Complex

Wilkinson, Barrie; Critchley, A.; Stirling, Colin J.

doi:10.1074/jbc.271.41.25590

Cited by 142 publications

(156 citation statements)

References 56 publications

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“…For example, the Saccharomyces cerevisiae Sec61p protein requires intramolecular cooperation between sequences located in the C terminus and upstream sequences in TM5 for correct membrane topogenesis (36). Similar signal sequences have been identified within the human P glycoprotein (29) and the cystic fibrosis transmembrane conductance regulator protein (28).…”

Section: Resultsmentioning

confidence: 89%

Reassessing the Role of Region A in Pit1-Mediated Viral Entry

Farrell

Russ

Murthy

et al. 2002

J Virol

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The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 ( Pit1 is the human ortholog of a ubiquitous multiple-membrane-spanning protein that functions as the type III sodium phosphate cotransporter (14, 19) and as the receptor for feline leukemia virus type B (FeLV-B) (33), woolly monkey virus (33), gibbon ape leukemia virus (GALV) (18), and 10A1 murine leukemia virus (10A1 MLV) (17, 38). Pit2, another type III sodium phosphate cotransporter, is the human ortholog of a highly related protein (approximately 60% amino acid identity), which functions as a receptor for amphotropic murine leukemia virus (17, 34), 10A1 MLV, and FeLV-B molecular recombinants (30), but not for GALV or naturally occurring FeLV-B isolates (21). The ability of Pit1 and Pit2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to identify regions within Pit1 important for virus receptor function implicated residues 550 to 558 for both GALV and FeLV-B entry. It has been proposed that this nine-Pit1-residue stretch, designated region A, is the binding site for both of these viruses (6, 11, 35) based on the following observations: (i) mutations in Pit1 region A render Pit1 nonfunctional for GALV or FeLV-B entry (3, 9, 22, 27, 31, 32); (ii) Pit2, MusPit1 (the murine ortholog of Pit1), and Pho-4 (a distantly related Neurospora crassa phosphate transporter), which are not GALV or FeLV-B receptors, become functional receptors when region A residues are substituted for the corresponding residues of these proteins (12, 13, 33); and (iii) region A is predicted by Kyte-Doolittle hydropathy plots to reside in an extracellular domain (12). It should be noted, however, that all published reports supporting the role of region A as a binding site have been based on functional assays of viral entry rather than actual receptor-binding studies (reviewed in reference 20). Therefore we sought to determine if mutations in region A that abolish virus receptor function do so by abolishing virus binding.We have used data derived from the analyses of epitopetagged Pit proteins, glycosylation studies, and transmembrane (TM) structure predictions performed by using the PHD PredictProtein algorithm (23, 24) to derive a...

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Section: Resultsmentioning

confidence: 89%

Reassessing the Role of Region A in Pit1-Mediated Viral Entry

Farrell

Russ

Murthy

et al. 2002

J Virol

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“…The observation that the ⌬NGGLQP mutation had a prominent effect on the topology of small Pgp fragments but had little effect on the topology of full-length MDR1-Pgp protein indicates that other regions of MDR1-Pgp might influence the final topology of TM7a/b and TM8 in the mature protein. Such a phenomenon has been observed during Sec61p topogenesis in yeast (Wilkinson et al, 1996). It is clear from these studies that both local and distant structural Figure 10.…”

Section: Molecular Biology Of the Cell 2694mentioning

confidence: 82%

“…Alternatively, native polytopic proteins may acquire their topology through cooperative and/or synergistic interactions between multiple topogenic determinants (Calamia and Manoil, 1992;Wilkinson et al, 1996;Lu et al, 1997). This latter process involves transient uncoupling of translocation and membrane integration events Lin and Addison, 1995), posttranslational translocation of peptide loops (Lu et al, 1997), or even posttranslational reorientation of TM helices (Wilkinson et al, 1996). Thus recent studies have added new complexities to the traditional view of polytopic protein biogenesis by demonstrating that a given TM topology may be generated through several alternate combinations of topogenic events.…”

Section: Introductionmentioning

confidence: 99%

“…In the simplest case, this process proceeds cotranslationally at the ER membrane as independent signal (anchor) and ST sequences direct sequential rounds of translocation initiation, translocation termination, and membrane integration (Rothman et al, 1988;Wessels and Spiess, 1988;Lipp et al, 1989;Shi et al, 1995). Alternatively, native polytopic proteins may acquire their topology through cooperative and/or synergistic interactions between multiple topogenic determinants (Calamia and Manoil, 1992;Wilkinson et al, 1996;Lu et al, 1997). This latter process involves transient uncoupling of translocation and membrane integration events Lin and Addison, 1995), posttranslational translocation of peptide loops (Lu et al, 1997), or even posttranslational reorientation of TM helices (Wilkinson et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

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Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Moss

Helm

et al. 1998

MBoC

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Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single-and a doublespanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8. INTRODUCTIONTransmembrane (TM) 1 topology of most eukaryotic integral membrane proteins is established at the endoplasmic reticulum (ER) membrane as specific topogenic determinants (e.g., signal, stop transfer [ST], and signal anchor sequences) emerge from the ribosome and engage their cognate cytosolic and/or membrane bound receptors (Blobel, 1980). This process of topogenesis involves at least four distinct steps: 1) ER membrane targeting, 2) translocation initiation, 3) translocation termination, and 4) membrane integration (reviewed in Rapoport et al., 1996;Johnson, 1997). Topogenic determinants in naturally occurring proteins usually direct these events efficiently and completely, thus ensuring a single uniform topology for any given cohort of nascent polypeptide chains. Failure to complete one or more of these steps, however, may result in proteins with heterogeneous topological outcomes. For example, mutagenesis studies of ST (Kuroiwa et al., 1991) and signal anchor (Parks et al., 1989;Parks and Lamb, 1991) sequences have shown that under certain conditions, topogenic determinants may generate mixed populations of nascent chains exhibiting secretory, N-trans (type I) and/or C-trans (type II) TM topology. Specialized topogenic determinants in naturally occurring proteins may also direct alternate topologies. In the case of murine plasminogen activator inhibitor 2, cytosolic and secretory iso-* Corresponding author. Present address: Division of Molecular Medicine, Oregon Health Sciences University, Portland, OR 97201-3098. 1 Abbreviations used: ER, endoplasmic reticulum; MDR1-Pgp, human P-glycoprotein; Pgp, P-glycoprotein; PK, proteinase K; ST, s...

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“…This channel in the ER membrane of both yeast and mammalian cells is formed by the heterotrimeric Sec61 complex, which consists of Sec61p (Sec61␣ in mammals), Sbh1p (Sec61␤), and Sss1p (Sec61␥). Sec61p is an essential polytopic protein with 10 transmembrane domains that line the protein-conducting channel in the ER membrane (16). A nonessential homologue of Sec61p, Ssh1p, forms a complex homologous to the Sec61 complex containing the Sbh1p homologue Sbh2p and Sss1p (17); the Ssh1 complex may have a specialized role in cotranslational translocation into the yeast ER, but Ssh1p is not required for misfolded protein export from the ER (15,17).…”

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confidence: 99%

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane

Gillece

Pilon

Römisch

2000

Proc. Natl. Acad. Sci. U.S.A.

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Peptides and misfolded secretory proteins are transported efficiently from the endoplasmic reticulum (ER) lumen to the cytosol, where the proteins are degraded by proteasomes. Protein export depends on Sec61p, the ribosome-binding core component of the protein translocation channel in the ER membrane. We found that prebinding of ribosomes abolished export of a glycopeptide from yeast microsomes. Deletion of SSH1, which encodes a ribosomebinding Sec61p homologue in the ER, had no effect on glycopeptide export. A collection of cold-sensitive sec61 mutants displayed a variety of phenotypes: two mutants strongly defective in misfolded protein export from the ER, sec61-32 and sec61-41, displayed only minor peptide export defects. Glycopeptide export was severely impaired, however, in several sec61 mutants that were only marginally defective in misfolded protein export. In addition, a mutation in SEC63 strongly reduced peptide export from the ER. ER-luminal ATP was required for both misfolded protein and glycopeptide export. We conclude that the protein translocation channel in the ER membrane mediates glycopeptide transport across the ER membrane.

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Determination of the Transmembrane Topology of Yeast Sec61p, an Essential Component of the Endoplasmic Reticulum Translocation Complex

Cited by 142 publications

References 56 publications

Reassessing the Role of Region A in Pit1-Mediated Viral Entry

Reassessing the Role of Region A in Pit1-Mediated Viral Entry

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane

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