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, Jürgen; Schmidt, Daniel R.; Glavy, Joseph S.; Blobel, Günter; Schwartz, Thomas

doi:10.1074/jbc.c300180200

Cited by 44 publications

(40 citation statements)

References 26 publications

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“…In the same study, those investigators showed that expression of a dominantnegative GEF of GBF1 (which can interact with ARF1) also led to dissolution of the trans-Golgi and ATP7A-containing compartments, although copper-responsive trafficking was lost. The Sec61␤ yeast homologs sbh1p and sbh2p were shown in a different study to function as GEFs, consistent with the observation that they contain homologous sec7 domains required for GEF function (Helmers et al, 2003). Our data suggest that Sec61␤ may function as a GEF and may modulate the ATP7A trans-Golgi compartment in a manner similar to that of the yeast GEF GBF1, possibly in concert with the ARF family of proteins.…”

Section: Discussionsupporting

confidence: 70%

Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

Abada¹,

Larson

Manorek

et al. 2012

Mol Pharmacol

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The Sec61 protein translocon is a multimeric complex that transports proteins across lipid bilayers. We discovered that the Sec61␤ subunit modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. To investigate the mechanism, expression of Sec61␤ was constitutively knocked down in 2008 ovarian cancer cells. Sec61␤ knockdown (KD) resulted in 8-, 16.8-, and 9-fold resistance to cisplatin (cDDP), carboplatin, and oxaliplatin, respectively. Sec61␤ KD reduced the cellular accumulation of cDDP to 67% of that in parental cells. Baseline copper levels, copper uptake, and copper cytotoxicity were also reduced. Because copper transporters and chaperones regulate platinum drug accumulation and efflux, their expression in 2008 Sec61␤-KD cells was analyzed; ATP7A was found to be 2-to 3-fold overexpressed, whereas there was no change in ATP7B, ATOX1, CTR1, or CTR2 levels. Cells lacking ATP7A did not exhibit increased cDDP resistance upon knockdown of Sec61␤. Sec61␤-KD cells also exhibited altered ATP7A cellular distribution. We conclude that Sec61␤ modulates the cytotoxicity of many chemotherapeutic agents, with the largest effect being on the platinum drugs. This modulation occurs through effects of Sec61␤ on the expression and distribution of ATP7A, which was shown previously to control platinum drug sequestration and cytotoxicity.

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Section: Discussionsupporting

confidence: 70%

Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

Abada¹,

Larson

Manorek

et al. 2012

Mol Pharmacol

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“…Sec61 has been copurified with Sec66 as part of a posttranslational translocation complex that includes Sec63 and Sec72 (34). Srp102 is part of the receptor complex for the signal recognition particle that targets ribosomes harboring secreted or membrane proteins to the Sec61 complex, and independent lines of evidence indicate it is closely associated with Sec61 components (35,36).…”

Section: Resultsmentioning

confidence: 99%

Large-scale identification of yeast integral membrane protein interactions

Miller

Lo²,

Ben‐Hur³

et al. 2005

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

257

205

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We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions.Saccharomyces cerevisiae ͉ split-ubiquitin ͉ support vector machine S ystematic studies of protein interactions in yeast have provided insights into the functions of many of the proteins encoded by this single-celled eukaryote. However, the roles of many integral membrane proteins remain poorly understood. Biochemical purifications require detergents to isolate proteins away from lipid molecules, and the large-scale nature of the affinity precipitation͞mass spectrometry projects (1, 2) precluded adjusting the detergents for individual integral membrane proteins. Two-hybrid assays (3, 4) require that the two proteins localize to the nucleus; integral membrane proteins, targeted to an aqueous nuclear environment, may aggregate or misfold.To increase the representation of integral membrane proteins in the protein-protein interaction network of Saccharomyces cerevisiae, we examined pair-wise interactions among 705 integral membrane proteins by the split-ubiquitin membrane yeast two-hybrid system (5). This modified form of the split-ubiquitin assay (6-8) is one of several hybrid protein approaches that detect interactions occurring at membranes. The split-ubiquitin membrane yeast two-hybrid system allows direct identification of yeast transformants that encode a pair of interacting proteins by use of a transcriptional reporter.Analyses of previous large-scale interaction data sets revealed significant numbers of false negatives and false positives (9). False negatives may represent interactions unsuitable for detection by a particular technique and, thus, may not be easily remedied. False positives can potentially be identified by a failure to be validated through additional experiments. However, the large-scale nature of this study and the difficulties associated with biochemical analysis of integral membrane proteins preclude confirmation of these results by alternative experimental approaches. Therefore, we used a learning algorithm, the support vector machi...

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“…5 and 6). Previously a segment in the Sbh1p cytosolic domain was shown to display homology with the Sec7 domain responsible for guanine nucleotide exchange activity for ADP-ribosylation factor family GTPases and it was shown that Sbh1p can act as a guanine nucleotide exchange factor for signal recognition particle receptor ␤ (14). The Sec61 ␤ subunit was also shown to interact with the large ribosomal subunit and was proposed to mediate ribosome docking with the Sec61 complex (13).…”

Section: Discussionmentioning

confidence: 99%

“…The ␤ subunit is not essential but has a facilitating role in translocation in mammalian cells (12). In addition to its interactions with Sec61p, the mammalian ␤ subunit has been shown to interact with signal peptidase (12), and the ribosome (13) and the yeast ␤ subunit, Sbh1p, was shown to act as a guanine nucleotide exchange factor for signal recognition particle receptor (14). The two yeast ␤ subunits, Sbh1p and Sbh2p (previously also called Seb1p and Seb2p), are encoded by non-essential genes.…”

mentioning

confidence: 99%

The Transmembrane Domain Is Sufficient for Sbh1p Function, Its Association with the Sec61 Complex, and Interaction with Rtn1p

Feng

Zhao

Soromani

et al. 2007

Journal of Biological Chemistry

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The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The ␣-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored ␤ subunit Sbh1p, its close homologue Sbh2p, and the ␥ subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1⌬ sbh2⌬ cells, whereas there is a limited reduction of post-translational tranlocation and no effect on export of a mutant form of ␣-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1⌬ sbh2⌬ cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61␤ homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.

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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

Cited by 44 publications

References 26 publications

Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

Large-scale identification of yeast integral membrane protein interactions

The Transmembrane Domain Is Sufficient for Sbh1p Function, Its Association with the Sec61 Complex, and Interaction with Rtn1p

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