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R Schekman

Publications and source records attributed to R Schekman.

At least 109 records · Page 6Linked to original sources
PubMed1989

Clathrin: a role in the intracellular retention of a Golgi membrane protein.

Yeast mutants deficient in the clathrin heavy chain secrete a precursor form of the alpha-factor, a peptide-mating pheromone. Analysis of this defect indicates that the endoprotease Kex2p, which is responsible for initiating proteolytic maturation of the alpha-factor precursor in the Golgi apparatus, is unexpectedly present at the plasma membrane in mutant cells. This result suggest that clathrin is required for the retention of Kex2p in the Golgi apparatus.

Cell Compartmentation↗
PubMed1989

Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro.

The SEC23 gene product (Sec23p) is required for transport of secretory, plasma membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex in Saccharomyces cerevisiae. Molecular cloning and biochemical characterization demonstrate that Sec23p is an 84 kd unglycosylated protein that resides on the cytoplasmic surface of a large structure, possibly membrane or cytoskeleton. Vigorous homogenization of yeast cells or treatment of yeast lysates with reagents that desorb peripheral membrane proteins releases Sec23p in a soluble form. Protein transport from the endoplasmic reticulum to the Golgi in vitro depends upon active Sec23p. Thermosensitive transport in sec23 mutant lysates is restored to normal when a soluble form of wild-type Sec23p is added, providing a biochemical complementation assay for Sec23p function. Gel filtration of yeast cytosol indicates that functional Sec23p is a large oligomer or part of a multicomponent complex.

Amino Acid Sequence↗
PubMed1989

Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation.

The role of the SEC7 gene product in yeast intercompartmental protein transport was examined. A spectrum of N-linked oligosaccharide structures, ranging from core to nearly complete outer chain carbohydrate, was observed on glycoproteins accumulated in secretion-defective sec7 mutant cells. Terminal alpha 1-3-linked outer chain mannose residues failed to be added to N-linked glycoproteins in sec7 cells at the restrictive temperature. These results suggest that outer chain glycosyl modifications do not occur within a single compartment. Additional evidence consistent with subdivision of the yeast Golgi apparatus came from a cell-free glycoprotein transport reaction in which wild-type membranes sustained outer chain carbohydrate growth up to, but not including, addition of alpha 1-3 mannose residues. Golgi apparatus compartments may specialize in addition of distinct outer chain determinants. The SEC7 gene product was suggested to regulate protein transport between and from functional compartments of the yeast Golgi apparatus.

Biological Transport↗
PubMed1989

Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast.

Genes that function in translocation of secretory protein precursors into the ER have been identified by a genetic selection for mutant yeast cells that fail to translocate a signal peptide-cytosolic enzyme hybrid protein. The new mutants, sec62 and sec63, are thermosensitive for growth and accumulate a variety of soluble secretory and vacuolar precursors whose electrophoretic mobilities coincide with those of the corresponding in vitro translated polypeptides. Proteolytic sensitivity of precursor molecules in extracts of mutant cells confirms that polypeptide translocation is blocked. Some form of interaction among the SEC61 (Deshaies, R. J., and R. Schekman. 1987. J. Cell Biol. 105:633-645), SEC62 and SEC63 gene products is suggested by the observation that haploid cells containing any pair of the mutations are inviable at 24 degrees C and show a marked enhancement of the translocation defect. The translocation defects of two mutants (sec62 and sec63) have been reproduced in vitro. sec63 microsomes display low and thermolabile translocation activity for prepro-alpha-factor (pp alpha F) synthesized with a cytosol fraction from wild type yeast. These gene products may constitute part of the polypeptide recognition or translocation apparatus of the ER membrane. Pulse-chase analysis of the translocation-defective mutants demonstrates that insertion of pp alpha F into the ER can proceed posttranslationally.

Cloning, Molecular↗
PubMed1989

SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.

Amino Acid Sequence↗
PubMed1989

Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae.

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

Amino Acid Sequence↗
PubMed1988

SEC7 encodes an unusual, high molecular weight protein required for membrane traffic from the yeast Golgi apparatus.

Saccharomyces cerevisiae with mutations at the sec7 locus are pleiotropically deficient in protein transport within the Golgi apparatus and proliferate a large array of Golgi cisternae at a restrictive growth temperature (37 degrees C). The SEC7 gene and its product (Sec7p) have been evaluated by molecular cloning and sequence analysis. Two genes that allow sec7 mutant cells to grow at 37 degrees C are represented in wild-type yeast DNA libraries. A single copy of the authentic SEC7 gene permits growth of mutant cells, whereas the other gene suppresses growth deficiency only when expressed from a multicopy plasmid. The SEC7 gene is contained on a 8.4-kilobase pair SphI restriction fragment, portions of which hybridize to a single 6-kilobase pair mRNA. The gene is essential for yeast vegetative growth. DNA sequence analysis of this region detects a single open reading frame with the potential to encode a 2008-amino acid-long hydrophilic protein of 230 kDa. Putative Sec7p contains an unusual, highly charged acidic domain of 125 amino acids with 29% glutamate, 18% aspartate, and 21% serine. Within this region, stretches of 14 consecutive glutamate residues and 13 consecutive glutamates/aspartates are predicted. This domain in Sec7p may serve a structural role to interact with lipids or proteins on the cytoplasmic surface of the Golgi apparatus.

Amino Acid Sequence↗
PubMed1988

Reconstitution of SEC gene product-dependent intercompartmental protein transport.

Transport of alpha-factor precursor from the endoplasmic reticulum to the Golgi apparatus has been reconstituted in gently lysed yeast spheroplasts. Transport is measured through the coupled addition of outer-chain carbohydrate to [35S]methionine-labeled alpha-factor precursor translocated into the endoplasmic reticulum of broken spheroplasts. The reaction is absolutely dependent on ATP, stimulated 6-fold by cytosol, and occurs between physically separable sealed compartments. Transport is inhibited by the guanine nucleotide analog GTP gamma S. sec23 mutant cells have a temperature-sensitive defect in endoplasmic reticulum-to-Golgi transport in vivo. This defect has been reproduced in vitro using sec23 membranes and cytosol. Transport at 30 degrees C with sec23 membranes requires addition of cytosol containing the SEC23 (wild-type) gene product. This demonstrates that an in vitro inter-organelle transport reaction depends on a factor required for transport in vivo. Complementation of sec mutations in vitro provides a functional assay for the purification of individual intercompartmental transport factors.

Adenosine Triphosphate↗
PubMed1988

The yeast SEC53 gene encodes phosphomannomutase.

Yeast sec53 cells incubated at a restrictive temperature (37 degrees C) accumulate inactive and incompletely glycosylated forms of secretory proteins within the lumen of the endoplasmic reticulum. A defect in glycosylation of alpha-factor precursor has been reproduced in vitro using membranes and cytosol isolated from sec53 mutant cells. Normal glycosylation is restored in reactions supplemented with a cytosolic fraction from wild type cells, with GDP-mannose, or with mannose 1-phosphate and GTP, but not with mannose 6-phosphate and GTP. This pattern of stimulation suggests that extracts of sec53 cells are deficient in phosphomannomutase activity or in the production of a precursor of mannose 1-phosphate. Several lines of evidence demonstrate that SEC53 encodes the yeast phosphomannomutase. Direct assay of soluble fractions from independent alleles of sec53 shows low to negligible phosphomannomutase, but nearly normal levels of phosphomannoisomerase activity. The residual phosphomannomutase activity in mutant cell lysates is thermolabile in proportion to the severity of the sec53 cell growth defect. Introduction of the SEC53 gene on a multicopy plasmid into sec53 or wild type yeast and into Salmonella typhimurium results in an increase in phosphomannomutase activity that correlates with elevated expression of the Sec53 protein. Finally, the Sec53 protein and phosphomannomutase activity cofractionate exactly in a 70-fold partial purification involving gel filtration and DEAE chromatography. The secretory defect in sec53 cells may now be explained by a deficit in GDP-mannose production.

Enzyme Stability↗
PubMed1988

A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides.

Depletion of a subset of 70K stress proteins in yeast mutants shows that they are involved in the post-translational import of precursor polypeptides into both mitochondria and the lumen of the endoplasmic reticulum. The identification of such a basic function may explain the remarkable evolutionary conservation of the gene family encoding these proteins.

Biological Evolution↗
PubMed1988

Secretory vesicles externalize the major plasma membrane ATPase in yeast.

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.

Acid Phosphatase↗
PubMed1988

Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast.

Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.

Animals↗
PubMed1988

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast.

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.

Amino Acid Sequence↗
PubMed1988

Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway.

Immunoelectron microscopy of Saccharomyces cerevisiae cells embedded in Lowicryl K4M has been used to localize invertase and plasma membrane (PM) ATPase in secretory organelles. sec mutant cells incubated at 37 degrees C were prepared for electron microscopy, and thin sections were incubated with polyclonal antibodies, followed by decoration with protein A-gold. Specific labeling of invertase was seen in the lumen of the endoplasmic reticulum, Golgi apparatus, and secretory vesicles in mutant cells that exaggerate these organelles. PM ATPase accumulated within the same organelles. Double-immune labeling revealed that invertase and PM ATPase colocalized in secretory vesicles. These results strengthen the view that secretion and plasma membrane assembly are biosynthetically coupled in yeast.

Adenosine Triphosphatases↗