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R Schekman

Publications and source records attributed to R Schekman.

At least 73 records · Page 4Linked to original sources
PubMed1994

Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway.

Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy.

Cell Nucleus↗
PubMed1994

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↗
PubMed1993

"BFA bodies": a subcompartment of the endoplasmic reticulum.

A specialized region of the endoplasmic reticulum--the BFA body--is defined by the site of accumulation of coatomer when nonclathrin coat protein (COP)-coated vesicle assembly is prevented by the drug brefeldin A (BFA). BFA bodies are formed by part smooth, part rough domains of endoplasmic reticulum that are cis to the classical transitional endoplasmic reticulum and to BFA-induced Golgi remnants.

Animals↗
PubMed1993

SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER.

In yeast a type II integral membrane glycoprotein that is essential for transport vesicle budding from the endoplasmic reticulum (ER) is encoded by SEC12 (refs 1-3). SAR1 was discovered as a multicopy suppressor of the sec12-1ts strain and encodes a GTPase of M(r) 21,000 (21K) also essential for vesicle budding from the ER. Sar1 is a peripherally associated membrane protein which shows enhanced membrane binding in cells containing elevated levels of Sec12 protein (refs 6, 7). We show here that a purified fragment of Sec12 promotes guanine-nucleotide dissociation from Sar1 whereas the purified mutant Sec12-1 has only 15% of the wild-type activity. GTP hydrolysis by Sar1 is not enhanced by Sec12, but is stimulated more than 50-fold by a mixture of Sec12 and Sec23, a GTPase-activating protein specific for Sar1 (ref. 8). We propose that Sec12 catalyses Sar1 guanine-nucleotide exchange in a process that recruits Sar1 to a vesicle formation site on the ER membrane.

ADP-Ribosylation Factors↗
PubMed1993

Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum.

The binding and hydrolysis of guanosine triphosphate (GTP) by the small GTP-binding protein Sar1p is required to form transport vesicles from the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. Experiments revealed that an interaction between Sar1p and the Sec23p subunit of an oligomeric protein is also required for vesicle budding. The isolated Sec23p subunit and the oligomeric complex stimulated guanosine triphosphatase (GTPase) activity of Sar1p 10- to 15-fold but did not activate two other small GTP-binding proteins involved in vesicle traffic (Ypt1p and ARF). Activation of GTPase was inhibited by an antibody to Sec23p but not by an antibody that inhibits the budding activity of the other subunit of the Sec23p complex. Also, activation was thermolabile in pure samples of Sec23p that were isolated from two independent sec23 mutant strains. It appears that Sec23p represents a new class of GTPase-activating protein because its sequence shows no similarity to any known member of this family.

COP-Coated Vesicles↗
PubMed1993

Purification and characterization of SAR1p, a small GTP-binding protein required for transport vesicle formation from the endoplasmic reticulum.

SEC12 encodes an integral membrane glycoprotein essential for vesicle formation from the endoplasmic reticulum (ER) in yeast. The SAR1 gene was discovered as a multicopy suppressor of a sec12ts strain and encodes a 21-kDa GTP-binding protein also required for protein transport from the ER to the Golgi apparatus (Nakano, A., and Muramatsu, M. (1989) J. Cell Biol. 109, 2677-2691). We have purified Sar1p to apparent homogeneity from cells harboring a galactose-regulated recombinant SAR1. Purified Sar1p binds guanine nucleotides specifically and exhibits GTPase activity (0.001 min-1). Nucleotide exchange and hydrolysis rates are greatly increased in the presence of Mg2+ and nonionic detergents or phospholipids. An assay that measures the formation of a vesicle intermediate in ER to Golgi transport was devised that is dependent on the addition of purified Sar1p. This assay employs membranes prepared from wild-type cells and cytosol fractions depleted of Sar1p due to overproduction of Sec12p or by gel filtration chromatography. The gel-filtered cytosol requires the addition of Sar1p and GTP to support vesicle budding. Sar1p prebound with GTP gamma S inhibits Sar1p function in the vesicle formation assay. The results indicate a role for Sar1p in vesicle budding from the ER and suggest that GTP hydrolysis by Sar1p is required for this event.

Chromatography, Gel↗
PubMed1993

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↗
PubMed1993

Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.

Alleles↗
PubMed1993

A Sec63p-BiP complex from yeast is required for protein translocation in a reconstituted proteoliposome.

Reconstituted proteoliposomes derived from solubilized yeast microsomes are able to translocate a secreted yeast mating pheromone precursor (Brodsky, J. L., S. Hamamoto, D. Feldheim, and R. Schekman. 1993. J. Cell Biol. 120:95-107). Reconstituted proteoliposomes prepared from strains with mutations in the SEC63 or KAR2 genes are defective for translocation; the kar2 defect can be overcome by the addition of purified BiP (encoded by the KAR2 gene). We now show that addition of BiP to wild-type reconstituted vesicles increases their translocation efficiency three-fold. To identify other ER components that are required for translocation, we purified a microsomal membrane protein complex that contains Sec63p. We found that the complex also includes BiP, Sec66p (gp31.5), and Sec67p (p23). The Sec63p complex restores translocation activity to reconstituted vesicles that are prepared from a sec63-1 strain, or from cells in which the SEC66 or SEC67 genes are disrupted. BiP dissociates from the complex when the purification is performed in the presence of ATP gamma S or when the starting membranes are from yeast containing the sec63-1 mutation. We conclude that the purified Sec63p complex is active and required for protein translocation, and that the association of BiP with the complex may be regulated in vivo.

Adenosine Triphosphate↗
PubMed1993

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↗
PubMed1992

SEC21 is a gene required for ER to Golgi protein transport that encodes a subunit of a yeast coatomer.

Non-clathrin coated vesicles have been implicated in early steps of intercompartmental transport. A distinct set of coat proteins are peripherally associated with the exterior of purified mammalian intra-Golgi transport vesicles. The 'coatomer', a cytosolic complex containing a similar subunit composition to and sharing at least one subunit (beta-COP) with the coat found on vesicles, has been postulated to be the precursor of this non-clathrin coat. Here we describe the characterization of SEC21, an essential gene required for protein transport from the endoplasmic reticulum to the Golgi in the yeast Saccharomyces cerevisiae. The 105K product of this gene, Sec21p, participates in a cytosolic complex that we show to be a yeast homologue of the mammalian coatomer. These observations demonstrate that a non-clathrin coat protein plays an essential role in intercompartmental transport.

Amino Acid Sequence↗
PubMed1992

Distinct processes mediate glycoprotein and glycopeptide export from the endoplasmic reticulum in Saccharomyces cerevisiae.

Protein and peptide export from the Saccharomyces cerevisiae endoplasmic reticulum was examined in vitro using the secretory protein pro-alpha-factor and a synthetic tripeptide containing the acceptor site for N-linked glycosylation as substrates. The release of both glycosylated pro-alpha-factor and glycotripeptide from the endoplasmic reticulum was dependent on cytosol, temperature, and ATP. Antibodies against two proteins essential for the formation of transport vesicles, Sec23p and p105, inhibited glyco-pro-alpha-factor exit from the endoplasmic reticulum but did not affect the release of the glycosylated tripeptide. Furthermore, in contrast to pro-alpha-factor, the exported glycopeptide was not associated with a membrane fraction and did not acquire Golgi-specific alpha(1-6)-linked mannose residues. We conclude that the glycosylated tripeptide leaves the yeast endoplasmic reticulum by a route different from the secretory pathway, possibly through an ATP-driven pump.

Amino Acid Sequence↗
PubMed1992

Polypeptide translocation across the endoplasmic reticulum membrane.

Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition. Much of the excitement in the next few years will come from the resolution of current controversies. What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction. Another approach is to identify homologues of these molecules in S. cerevisiae and to assess their importance in in vivo translocation. Several mechanistic questions remain to be addressed as well. Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years.

Animals↗
PubMed1992

The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein.

The SEC17 gene of Saccharomyces cerevisiae is required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus. Here we report that the product of the SEC17 gene has the exact biochemical properties expected for a yeast homologue of the mammalian transport factor, alpha-SNAP. The DNA sequence of SEC17 codes for a protein of predicted molecular mass of 33 kDa. Immunoblotting indicates that Sec17p fractionates as a peripheral membrane protein and is mostly soluble when overexpressed, suggesting the presence of a saturable membrane receptor for Sec17p. Sec17p was purified from yeast cytosol using a SNAP-dependent in vitro mammalian Golgi transport assay. Kinetic analysis using this assay shows Sec17p acts temporally close to the fusion of transport vesicles with the medial Golgi compartment. In yeast extracts, Sec17p binds to Sec18p with a 1:1 stoichiometry. The interaction between Sec17p and Sec18p requires an activity provided by yeast membranes, and this putative membrane receptor activity is not extracted by high salt treatment of membranes.

Amino Acid Sequence↗
PubMed1992

Genetic and biochemical analysis of vesicular traffic in yeast.

Secretory, vacuolar and membrane protein transport in yeast occurs by processes that are highly conserved in eukaryotic cells. Recent years have seen a proliferation of approaches to the study of vesicular traffic, and in certain instances key breakthroughs have been achieved through the application of genetic and biochemical methods that are well suited to yeast as an experimental organism. The availability of the genetic approach has led to molecular insights concerning the mechanisms of vesicle biogenesis, targeting and fusion.

Biological Transport↗
PubMed1992

Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1992

Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.

Animals↗