Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.

Waters, M. Gerard; Blobel, Günter

doi:10.1083/jcb.102.5.1543

Cited by 310 publications

(174 citation statements)

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“…The precursors to a-factor and CPY, but not invertase, can be posttranslationally translocated across the membrane (Hansen et al, 1986;Rothblatt and Meyer, 1986;Waters and Blobel, 1986;Hansen and Walter, 1988). In this regard, we have previously demonstrated that the sec7l-1 and sec72-1 mutants accumulate ppCPY but not preinvertase .…”

Section: Discussionmentioning

confidence: 99%

“…Some preproteins can be translocated across the membrane, in a posttranslational manner in yeast (Hansen et al, 1986;Rothblatt and Meyer, 1986;Waters and Blobel, 1986). Posttranslational translocation of the precursor to a-factor is mediated by cytoplasmic heatshock proteins (Chirico et al, 1988;Deshaies et al, 1988) and cytoplasmic Ydjlp, a DnaJ homologue (Caplan et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

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Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

Fang

Green

1994

MBoC

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The sec7l-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1),\which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37°C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

Fang

Green

1994

MBoC

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“…A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated. The existence of a posttranslational reaction has allowed these investigators to demonstrate that protein translocation into the yeast ER is energy dependent (15,43,57). In addition, fractionation experiments suggest that the import reaction requires cytosolic components (58).…”

mentioning

confidence: 99%

“…The mechanism of protein permeation across the hydrophobic core of the ER membrane is not understood. Experiments with intermediates artificially blocked at various stages of membrane penetration suggest that this process is mediated by proteins, though they have yet to be identified by the existing assays (14).Recently, several groups have reconstituted protein translocation into the yeast ER in vitro (15,42,57). A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated.…”

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A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

Deshaies¹,

Schekman²

1987

The Journal of Cell Biology

404

233

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Abstract. We have devised a genetic selection for mutant yeast cells that fail to translocate secretory protein precursors into the lumen of the endoplasmic reticulum (ER). Mutant cells are selected by a procedure that requires a signal peptide-containing cytoplasmic enzyme chimera to remain in contact with the cytosol. This approach has uncovered a new secretory mutant, sec61, that is thermosensitive for growth and that accumulates multiple secretory and vacuolar precursor proteins that have not acquired any detectable posttranslational modifications associated with translocation into the ER. Preproteins that accumulate at the sec61 block sediment with the particulate fraction, but are exposed to the cytosol as judged by sensitivity to proteinase K. Thus, the sec61 mutation defines a gene that is required for an early cytoplasmic or ER membrane-associated step in protein translocation.T hE first step in the biogenesis of proteins destined for the secretory pathway is their insertion into the membrane of the endoplasmic reticulum (ER).I This process has been studied intensively in mammalian cells through the use of an in vitro assay that faithfully reproduces cotranslational translocation of secretory proteins into the lumen of the ER (2). Dissection of the components required for this activity has revealed the existence of both soluble and membrane-bound factors that participate in protein translocation. The signal recognition particle is a soluble ribonucleoprotein particle consisting of six polypeptides (54) and one molecule of 7SL RNA (55). The signal recognition particle binds to the signal sequence of a nascent preprotein (28, 56), thereby forming a complex that interacts with an integral membrane protein of the ER known as docking protein or signal recognition particle receptor (13,33). This targeting event is followed by cotranslational translocation of the preprotein into the ER lumen. Either during or shortly after the translocation event, the signal sequence is cleaved by the enzyme signal peptidase (10) and core oligosaccharides are transferred to specific asparagine residues (44) of the translocated polypeptide. The mechanism of protein permeation across the hydrophobic core of the ER membrane is not understood. Experiments with intermediates artificially blocked at various stages of membrane penetration suggest that this process is mediated by proteins, though they have yet to be identified by the existing assays (14).Recently, several groups have reconstituted protein translocation into the yeast ER in vitro (15,42,57). A yeast translation extract programmed with prepro-ct-factor mRNA di- rects the synthesis of an intact precursor which can insert co-or posttranslationaUy into yeast microsomes and become core-glycosylated. The existence of a posttranslational reaction has allowed these investigators to demonstrate that protein translocation into the yeast ER is energy dependent (15,43,57). In addition, fractionation experiments suggest that the import reaction requires cytosolic components (58). T...

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Yeast protein translocation complex: Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit

Toikkanen

Gatti

Takei

et al. 1996

Yeast

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A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion‐defective sec15–1 mutation. This gene encodes a protein homologous to the β subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane‐spanning region at the C‐terminus but no N‐terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61β subunit and 34% identity to the Arabidopsis thaliana Sec61β subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61–2 and sec61–3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N‐terminus exposed to the cytoplasm. The in vitro translated Seb1p was post‐translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature‐sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis. The sequences of SEB1 and SEB2 have been deposited in the EMBL database under Accession Numbers Z47789 and Z50012, respectively. The sequence of the Seb1 protein is identical to that of Sbh1p independently identified by Panzner et al. (Cell 81, 561–570. 1995) using a biochemical approach.

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Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.

Cited by 310 publications

References 34 publications

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.

Yeast protein translocation complex: Isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61β subunit

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