Recognition of a Subset of Signal Sequences by Ssh1p, a Sec61p-related Protein in the Membrane of Endoplasmic Reticulum of Yeast<i>Saccharomyces cerevisiae</i>

Wittke, Sandra; Dünnwald, Martin; Albertsen, Markus; Johnsson, Nils

doi:10.1091/mbc.01-10-0518

Cited by 52 publications

(50 citation statements)

References 47 publications

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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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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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“…Successful recombination was confirmed by diagnostic PCR and sequencing. P all N ub fusions, P CUP1 -SEC62-DHA, P CUP1 -SEC62 ∆C125 -DHA, P CUP1 -SEC62 ∆N144 -DHA, P CUP1 -SEC62 ∆C35 -DHA, and all signal-sequence-containing C ub -URA3 constructs were as described (Dünnwald et al, 1999;Wittke et al, 1999;Wittke et al, 2002). The C-terminal mutations of SEC63 were introduced into the P MET17 -SEC63-C ub -RURA3 vector by replacing the ClaI-SalI fragment spanning the 3′ end of SEC63 and the sequence connecting SEC63 with C ub by a ClaI, SalI cut PCR fragment containing the corresponding sequence but harboring the desired mutations.…”

Section: Construction Of Fusion Proteins Mutationsmentioning

confidence: 99%

“…All insertions were tested by diagnostic PCR and western blots of protein extracts obtained from the transformed strains. Functionality of the SEC63-ProA allele was confirmed by the Ura3p-based translocation assay (Wittke et al, 2002). The ProA fusions were detected by consecutive incubations with horseradish peroxidase coupled rabbit anti-goat and goat antirabbit antibodies (Biorad, Hercules, USA).…”

Section: Construction Of Fusion Proteins Mutationsmentioning

confidence: 99%

Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus

Wang

Johnsson

2005

Journal of Cell Science

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The heterotetrameric Sec62/63 complex associates with the heterotrimeric Sec61 complex to form the heptameric Sec complex. This complex is necessary and sufficient for post-translational protein translocation across the membrane of the endoplasmic reticulum. We show that Sec63p is phosphorylated at its C-terminal domain by the protein kinase CK2 and that this phosphorylation strengthens the interaction between the cytosolic domains of Sec63p and Sec62p. Exchanging either threonine 652 or threonine 654 against the nonphosphorylatable alanines in Sec63p impairs the binding to Sec62p and interferes with the efficient translocation of proteins across the membrane of the endoplasmic reticulum. These findings show that phosphorylation of Sec63p is required for tightly recruiting the putative signal-sequence-binding subunit Sec62p to the Sec complex.

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“…SDS-PAGE (polyacrylamide gel electrophoresis), transfer onto nitrocellulose and immunodetection of the transferred proteins was performed as described previously (Wittke et al, 2002). For sequential detection of proteins, membranes were stripped with 2% SDS and 100 mM β-mercaptoethanol for 30 minutes at 55°C.…”

Section: Gfp-skl Import Assaymentioning

confidence: 99%

Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome

Eckert

Johnsson

2003

Journal of Cell Science

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The protein import machinery of the peroxisome consists of many proteins,collectively called the peroxins. By applying the split-ubiquitin technique we systematically tested the pair-wise interactions between the Nub-and Cub-labeled peroxins for the first time in the living cells of the yeast Saccharomyces cerevisiae. We found that Pex10p plays a central role in the protein interaction network by connecting the ubiquitin conjugation enzyme Pex4p to the other members of the protein import machinery. A yeast strain harboring a deletion of PEX3 enabled us to estimate the influence of the peroxisomal membrane on the formation of a subset of the investigated protein-protein interactions.

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Recognition of a Subset of Signal Sequences by Ssh1p, a Sec61p-related Protein in the Membrane of Endoplasmic Reticulum of YeastSaccharomyces cerevisiae

Cited by 52 publications

References 47 publications

Large-scale identification of yeast integral membrane protein interactions

Large-scale identification of yeast integral membrane protein interactions

Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus

Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome

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