Nanotechnology. Flipping a molecular switch.
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D Feldheim
Publications and source records attributed to D Feldheim.
SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus.
The yeast SSS1 gene has been isolated as an extragenic high copy suppressor of sec61, a mutant displaying defects in protein translocation into the endoplasmic reticulum (ER). We found that SSS1 is an essential gene required for transfer of secretory precursors through the ER membrane. Here we demonstrate that the SSS1 product (Sss1p) is firmly bound to the ER membrane and exposes its amino-terminal half on the cytosolic side. Only detergent, or an alkali treatment, is effective at extracting Sss1p from the membrane. Coimmunoprecipitation experiments revealed that Sss1p and Sec61p participate in the same multisubunit complex. Cross-linking followed by immunoprecipitation specifically yielded an additional polypeptide of molecular mass 73 kDa. Moreover, Sss1p and Sec61p show mutually stabilizing interactions: Sss1p is destabilized in a sec61 mutant context, and mutated Sec61p is stabilized by Sss1p overproduction. These observations account for the isolation of SSS1 as a dosage-dependent suppressor of sec61. Since the polytopic integral membrane protein Sec61p is adjacent to translocating precursors and to ribosomes, and given the comparable translocation deficiencies of sss1 or sec61 mutants, we propose that Sss1p belongs to the "Sec61 subcomplex" that constitutes the pore of the membrane-bound translocation apparatus.
Biological Transport↗
Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex.
SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.
Amino Acid Sequence↗
Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70.
We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.
Biological Transport↗
Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.
SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.
Amino Acid Sequence↗
Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.
SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.
Amino Acid Sequence↗
Patients' reactions to house staff work rounds.
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