Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore

Crowley, Kathleen S.; Liao, Shuren; Worrell, Veronica; Reinhart, Gregory D.; Johnson, Arthur E.

doi:10.1016/0092-8674(94)90424-3

Cited by 327 publications

(263 citation statements)

References 36 publications

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“…The Sec61p-C 5 fragment consists of a fusion of residues NH 2 M1 3 F13-GS-L2323 M480COOH (264 amino acids). A 1.8-kbp HindIII fragment encoding Sec61p-C 5 was cloned into HindIIIlinearized pRS424 (42) to provide multicopy, TRP1-selectable expression of C 5 .…”

Section: Methodsmentioning

confidence: 99%

“…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: Methodsmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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“…For the simplest secretory proteins, when the signal sequence is recognized by the channel, the ribosome assumes a tight interaction with the channel that shields the protein from the cytoplasm (8, 10). Concomitantly, the channel opens toward the ER lumen, either via a conformational change in the channel or removal of a molecular plug covering the luminal aperture (11,12).Not all signal sequences initiate translocation in the same way. Beyond merely stimulating the initiation of translocation, signal sequences can regulate the association between the ribosome and translocon and the exposure of the nascent chain to the cytoplasm or ER lumen (13-16).…”

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confidence: 99%

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confidence: 99%

Signal Sequences Initiate the Pathway of Maturation in the Endoplasmic Reticulum Lumen

Rutkowski

Ott

Polansky

et al. 2003

Journal of Biological Chemistry

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An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated as well. Here we show that information encoded in the hydrophobic domain of a signal sequence influences the timing and efficiency of at least two steps in maturation, namely N-linked glycosylation and signal sequence cleavage. We demonstrate that these consequences correlate with and likely stem from the nature of the initial association made between the signal sequence and the translocon during the initiation of translocation. We propose a model by which these maturational events are controlled by the signal sequence-translocon interaction. Our work demonstrates that the pathway taken by a nascent chain through post-translational maturation depends on information encoded in its signal sequence.N-terminal signal sequences enable nascent secretory and transmembrane proteins to be targeted to the endoplasmic reticulum (ER) 1 for translocation. The signal sequence, newly emerged from the translating ribosome, is recognized in the cytoplasm by the signal recognition particle (1). By virtue of an interaction with its ER-localized receptor, signal recognition particle brings the ribosome-nascent chain complex to the ER membrane (2). Once at the ER, the ribosome is thought to facilitate the assembly or stabilization of the translocation channel, composed of the heterotrimeric Sec61 complex (3, 4). The 35-kDa polytopic Sec61␣ subunit appears to form the channel walls, whereas the smaller bitopic ␤-and ␥-subunits play an as yet undetermined role, perhaps in facilitating the insertion of the nascent chain into the channel (5-7).An important advance in the understanding of the initiation of translocation was the discovery of a second step of signal sequence recognition. In addition to being recognized by the signal recognition particle, the signal sequence also interacts with the translocation channel itself (8,9). Although the mechanism is not yet clear, this event is thought to stimulate the initiation of translocation. For the simplest secretory proteins, when the signal sequence is recognized by the channel, the ribosome assumes a tight interaction with the channel that shields the protein from the cytoplasm (8, 10). Concomitantly, the channel opens toward the ER lumen, either via a conformational change in the channel or removal of a molecular plug covering the luminal aperture (11,12).Not all signal sequences initiate translocation in the same way. Beyond merely stimulating the initiation of translocation, signal sequences can regulate the association between the ribosome and translocon and the exposure of the nascent chain to the cytoplasm or ER lumen (13-16). In at least one case, that of the prion protein, the consequence of this regulatory step is the governance of the ...

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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.…”

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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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Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore

Cited by 327 publications

References 36 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

Signal Sequences Initiate the Pathway of Maturation in the Endoplasmic Reticulum Lumen

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

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