The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation

Kalies, Kai‐Uwe; Rapoport, Tom A.; Hartmann, Enno

doi:10.1083/jcb.141.4.887

Cited by 124 publications

(124 citation statements)

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“…As a result the targeting machinery could not be linked to the occupied PCC. Sbh1p can be cross-linked to Spc25p, a subunit of the signal peptidase complex (25). Inaccessibility of the ␤-subunits of the PCCs may, therefore, not only be caused by the RNC complex binding to the PCCs, but also by the lateral recruitment within the plane of the ER membrane of other integral ER membrane proteins, such as the signal peptidase complex, the oligosaccharyltransferase or the additional subunits recruited to form the heptameric complex which is involved in posttranslational transport.…”

Section: Discussionmentioning

confidence: 99%

The β-Subunit of the Protein-conducting Channel of the Endoplasmic Reticulum Functions as the Guanine Nucleotide Exchange Factor for the β-Subunit of the Signal Recognition Particle Receptor

Helmers

Schmidt

Glavy

et al. 2003

Journal of Biological Chemistry

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Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the ␣-and ␤-subunits of the SRP receptor (SR). SR␤ is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SR␤-GTP in complex with the binding domain of SR␣ suggested that SR␤, like all ARF-type GTPases, requires a guanine nucleotide exchange factor (GEF) for function. Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the ␤-subunits of the two homologous heterotrimeric protein-conducting channels in yeast. Using a fluorescence nucleotide exchange assay, we show that the ␤-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SR␤. Both the cytosolic domain of Sec61␤ as well as the holo-Sec61␤, when part of the isolated trimeric Sec61p complex, function as the GEF for SR␤, whereas the same Sec61␤, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SR␤.Of the GTPases involved in cotranslational protein transport, the signal recognition particle (SRP) 1 subunit SRP54 recognizes and binds to the signal sequence of the nascent chain, presented by the ribosome nascent chain complex (RNC) (1). The interaction between SRP54 and the membrane bound SRP receptor (SR) selectively targets the RNC-SRP complex to the membrane of the endoplasmic reticulum (ER). This is followed by the release of the signal sequence from SRP54 and its insertion into the protein-conducting channel (PCC) that is formed by the oligomeric assembly of the trimeric Sec61p complex (2). The nascent chain translocates into the ER lumen, while the ribosome remains attached to the PCC.The GTPase domains of SRP54 and SR␣ are very similar in their structure and function. Structural studies of their prokaryotic homologues have shown that they form their own family within the GTPase superfamily (3, 4). They have low affinity for nucleotide and are both stable in their empty states (5, 6). SRP54 binds GTP when in contact with the RNC (6). The interaction between SRP54 and SR␣ leads to the formation of a GTP stabilized SRP-SR␣ complex (7). GTP hydrolysis dissociates the SRP-SR␣ complex (8), whereby SRP54 and SR␣ serve as mutual GTPase-activating proteins (GAP). This reciprocally symmetric interaction is unique among known GTPases (9).SR␤ is closely related to members of the ADP-ribosylation factor (ARF) GTPase family (10, 11). ARFs are conserved in all eukaryotes and are involved in the regulation of vesicle transport (12). SR␤ is present only in eukaryotes and contains a transmembrane segment providing the membrane anchor for SR␣ (13). However, a truncated protein representing the cytosolic GTPase domain without the transmembrane domain is functional in protein targeting (14). The function of the GTPase domain of SR␤ has long been a matter ...

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Section: Discussionmentioning

confidence: 99%

The β-Subunit of the Protein-conducting Channel of the Endoplasmic Reticulum Functions as the Guanine Nucleotide Exchange Factor for the β-Subunit of the Signal Recognition Particle Receptor

Helmers

Schmidt

Glavy

et al. 2003

Journal of Biological Chemistry

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“…The ratio of Golgi-to-ER staining seemed similar both by YFP fluorescence and anti-GFP antibody, arguing against an ER pool of misfolded chimera. A CFP-labeled Sec61␤ (Rolls et al, 1999), a translocon component localized to the ER (Kalies et al, 1998;Rolls et al, 1999), was then expressed to obtain a double-stable cell line. This construct localized to the ER, including the nuclear envelope, as judged by CFP fluorescence.…”

Section: The Generation Of a Cell Line Expressing Fluorescent Golgi Amentioning

confidence: 99%

Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

Axelsson

Warren

2004

MBoC

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Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes. INTRODUCTIONThe status of the Golgi apparatus as an organelle has been the subject of a long-running debate that focused initially on the route taken by transiting cargo (cisternal maturation or vesicle-mediated transport; Glick and Malhotra, 1998;Pelham and Rothman, 2000) and, more recently, on the mechanisms of duplication and partitioning that underlie its biogenesis (Marsh and Howell, 2002;Munro, 2002). If the Golgi apparatus is responsible for its biogenesis, then it can be viewed as an autonomous organelle (Pelletier et al., 2002). If, on the other hand, it depends on the endoplasmic reticulum, then it can be viewed as a dependent organelle, which exists only as a consequence of ER functioning (Zaal et al., 1999;Bevis et al., 2002;Munro, 2002).Golgi partitioning during mitosis in animal cells has been particularly controversial, with models ranging from partitioning by Golgi elements themselves (Lucocq et al., 1989;Misteli and Warren, 1995;Jesch and Linstedt, 1998;Shima et al., 1998;Jesch et al., 2001;Jokitalo et al., 2001) to the partial or even complete merger of the Golgi with the ER, which then mediates the partitioning process (Thyberg and Moskalewski, 1992;Zaal et al., 1999;Kano et al., 2000;Terasaki, 2000). Although most biochemical experiments have yielded consistent results, suggesting a separation of the ER and Golgi during mitosis (Jesch and Linstedt, 1998;Farmaki et al., 1999;Jesch et al., 2001), microscopic experiments have often yielded contradictory results (Thyberg and Moskalewski, 1992;Shima et al., 1998;Zaal et al., 1999;Jokitalo et al., 2001).A particular issue has been the Golgi haze observed by fluorescence microscopy of mitotic cells. Breakdown and fragmentation of the Golgi ribbon a...

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“…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)(6)(7).…”

mentioning

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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The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation

Cited by 124 publications

References 21 publications

The β-Subunit of the Protein-conducting Channel of the Endoplasmic Reticulum Functions as the Guanine Nucleotide Exchange Factor for the β-Subunit of the Signal Recognition Particle Receptor

The β-Subunit of the Protein-conducting Channel of the Endoplasmic Reticulum Functions as the Guanine Nucleotide Exchange Factor for the β-Subunit of the Signal Recognition Particle Receptor

Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

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

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