The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation

Crowley, Kathleen S.; Reinhart, Gregory D.; Johnson, Arthur E.

doi:10.1016/0092-8674(93)90640-c

Cited by 268 publications

(212 citation statements)

References 65 publications

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“…The potential importance of this putative amphipathic structure is underlined by its conservation both in Sec61␣ (19) and in a Schizosaccharomyces pombe homologue. 4 Our data indicate that both HS2 and HS5 require C-terminal sequences for their topogenesis. In the case of HS2, the Cterminal fusion data clearly report that neither HS2 nor HS3 span the bilayer.…”

Section: Discussionmentioning

confidence: 94%

“…Electrophysiological studies indicate the presence of large aqueous channels in rough ER membranes (3), and fluorophores incorporated into a translocating polypeptide report an aqueous environment when trapped within the membrane (4,5). Considerable support for the proposal has been provided by the identification of proteins located in proximity to translocating nascent polypeptides by use of cross-linking techniques (6).…”

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confidence: 99%

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

Wilkinson

Critchley

Stirling

1996

Journal of Biological Chemistry

142

147

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Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a proteinconducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.

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Section: Discussionmentioning

confidence: 94%

mentioning

confidence: 99%

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

Wilkinson

Critchley

Stirling

1996

Journal of Biological Chemistry

142

147

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“…For example, if TM7b terminated translocation, then TM8 would likely emerge from the ribosome in contact with cytosol (Liao et al, 1997). In chains where TM7b failed to terminate translocation, however, TM8 would emerge from the ribosome on an actively translocating chain (presumably moving through the Sec61 translocation channel [Borel and Simon, 1996;Laird and High, 1997] and shielded from the cytosol by the ribosome-membrane junction [Crowley et al, 1993[Crowley et al, , 1994). We therefore tested the topogenic behavior of TM8 using two chimeric proteins that mimic these potential presentations of TM8 to the ER by TM7a/b.…”

Section: Tm8 Directs Two Different Membrane-spanning Orientationsmentioning

confidence: 99%

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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“…The authenticity of such intermediates can be tested by releasing the halt with puromycin. This experimental system, in combination with positionspecific modification of nascent chain amino acids by crosslinking or fluorescence probes, allows detailed examination of the nascent chain environment, such as proteins in the proximity of the translocating polypeptide (Mothes et al 1994) or the hydropathic nature of the environment that the residues encounter during translocation (Crowley et al 1993). Finally, 'real biochemistry', such as purification of the machinery components and their reconstitution into proteoliposomes of defined composition, has now been achieved both in the E. coli and the ER systems Douville et al 1995;Görlich & Rapoport 1993;Panzner et al 1995).…”

Section: Introductionmentioning

confidence: 99%

The major pathways of protein translocation across membranes

Ito

1996

Genes to Cells

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The initial events in the localization of cell surface proteins include targeting of precursor molecules to the membrane and subsequent translocation across the membrane. The translocation reaction is mediated through a series of molecular interactions involving a number of protein factors. The trimeric SecY/Sec61 core complex, which is conserved throughout evolution, provides a proteinaceous pathway for translocation. The driving force for translocation is provided by the dynamic motion of the SecA ATPase in prokaryotes and by polypeptide elongation through the ribosome-Sec61 junction in eukaryotes.

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The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation

Cited by 268 publications

References 65 publications

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

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

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

The major pathways of protein translocation across membranes

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