Search PubMed⌕ Search

Biomedical subjects

R Schekman

Publications and source records attributed to R Schekman.

At least 127 records · Page 7Linked to original sources
PubMed1987

Isolation of secretory vesicles from Saccharomyces cerevisiae.

Purification of secretory vesicles from Saccharomyces cerevisiae has been hindered because these organelles normally represent a small proportion of cellular membranes. In the yeast secretory mutant sec1, secretory vesicles accumulate intracellularly in large quantities. Using a sec1 strain we have devised a procedure for the partial purification of these vesicles. The purification employs differential and density gradient centrifugations and an electrophoretic separation of membranes. The fractions obtained from this procedure are enriched for secretory vesicles at least fivefold over other cellular membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane fractions reveals a distinct set of polypeptides associated with secretory vesicles.

Acid Phosphatase↗
PubMed1987

Product of SEC53 is required for folding and glycosylation of secretory proteins in the lumen of the yeast endoplasmic reticulum.

Yeast secretory mutant sec53 cells accumulate inactive secretory glycoprotein precursors that remain associated with the endoplasmic reticulum (ER) at the restrictive temperature (37 degrees C). The possibility that precursor polypeptides fail to penetrate completely into the ER lumen was tested by examining the protease accessibility of accumulated invertase, mating pheromone precursor prepro-alpha-factor and the vacuolar protein precursor procarboxypeptidase Y in cell lysates. In all three cases, the secretory protein precursors are protected from the action of exogenous protease unless the membrane is permeabilized by including Triton X-100 or saponin in the incubation. These results suggest that the sec53 defect allows complete polypeptide translocation. Consistent with this interpretation, the precursor of invertase accumulates in a signal peptide-processed form. In addition, invertase and prepro-alpha-factor precursors contain a small amount of possibly aberrant carbohydrate. In mutant cells or in wild type cells treated with tunicamycin, a 10-kDa fragment of the N terminus of mature invertase assumes a conformation that is resistant to trypsin with or without detergent. This domain may be associated with an ER protein or may simply assume an unusual conformation as a consequence of deficient glycosyl modification.

Carboxypeptidases↗
PubMed1987

Structure, assembly, and secretion of octameric invertase.

Yeast invertase forms a homo-octamer of core glycosylated subunits during assembly in the lumen of the endoplasmic reticulum. This form has been purified from mutant cells (sec18) in which transport of secreted proteins from the endoplasmic reticulum is blocked. No heterologous protein subunits are found in the purified material. Analysis of invertase derived from wild type cells or from mutant cells blocked at subsequent stages in secretion demonstrates that invertase remains a homo-octamer throughout the pathway even though the extent of subunit glycosylation increases. Purified octameric invertase is dissociated into dimer units that reassociate in the presence of polyethylene glycol. Negatively stained preparations show the dissociated enzyme as individual spheres, whereas octameric invertase appears as four associated spheres. Assembly of the octamer in vitro and in vivo is facilitated by the presence of N-linked carbohydrate. Selective release of dimeric glycosylated invertase from intact yeast cells suggests that oligomerization helps retain the enzyme in the periplasmic space.

Acid Phosphatase↗
PubMed1987

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

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.

Endoplasmic Reticulum↗
PubMed1987

Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae.

Clathrin is important but not essential for yeast cell growth and protein secretion. Diploid Saccharomyces cerevisiae cells heterozygous for a clathrin heavy-chain gene (CHC1) disruption give rise to viable, slow-growing, clathrin heavy-chain-deficient meiotic progeny (G. Payne and R. Schekman, Science 230:1009-1014, 1985). The possibility that extragenic suppressors account for growth of clathrin-deficient cells was examined by deletion of CHC1 from haploid cell genomes by single-step gene transplacement and independently by introduction of a centromere plasmid carrying the complete CHC1 gene into diploid cells before eviction of a chromosomal CHC1 locus and subsequent tetrad analysis. Both approaches yielded clathrin-deficient haploid strains. In mutants missing at least 95% of the CHC1 coding domain, transcripts related to CHC1 were not detected. The time course of invertase modification and secretion was measured to assess secretory pathway functions in the viable clathrin-deficient cells. Core-glycosylated invertase was converted to the mature, highly glycosylated form at equivalent rates in mutant and wild-type cells. Export of mature invertase from mutant cells was delayed but not prevented. Abnormal vacuoles, accumulated vesicles, and Golgi body-derived structures were visualized in mutant cells by electron microscopy. We conclude that extragenic suppressors do not account for the viability of clathrin-deficient cells and, furthermore, that many standard laboratory strains can sustain a CHC1 disruption. Clathrin does not appear to mediate protein transfer from the endoplasmic reticulum to the Golgi body but may function at a later stage of the secretory pathway.

Clathrin↗
PubMed1986

Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae.

Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells. Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a "metabolic starvation" protocol. Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period. The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation. Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts. The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.

Cell Cycle↗
PubMed1986

Interorganelle transfer and glycosylation of yeast invertase in vitro.

Core glycosylated proteins formed in the yeast endoplasmic reticulum (ER) are transported to the Golgi body, where oligosaccharides are elongated by addition of outer-chain carbohydrate. The transport process is blocked in a temperature-sensitive secretion mutant (sec18) of Saccharomyces cerevisiae, which accumulates core glycosylated invertase (product of SUC2; EC 3.2.1.26) in the ER. To approach the molecular mechanism of this transport process, we have devised a reaction in which core glycosylated invertase, accumulated in sec18 cells, is transferred to the Golgi body in vitro. For this purpose, membranes from sec18, SUC2 cells that are also defective in an outer chain alpha-1----3-mannosyltransferase (mnnl) are mixed with membranes from a strain that contains the transferase but is deficient in invertase (MNNl, delta SUC2). Transfer is detected by the acquisition of outer-chain alpha-1----3-linked mannose residues dependent on both donor and recipient membranes. The reaction is temperature and detergent sensitive and requires ATP, GDP-mannose, Mg2+, and Mn2+, and the product invertase remains associated with sedimentable membranes. Treatment of donor, but not acceptor, membranes with N-ethylmaleimide or trypsin inactivates transfer competence. These characteristics suggest that the ER, or a vesicle derived from the ER, contributes invertase to a chemically distinct compartment where mannosyl modification is executed.

Biological Transport↗
PubMed1986

Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y.

The structural gene for yeast vacuolar carboxypeptidase Y (PRC1) has been cloned by complementation of the prc1-1 mutation. As much as an eightfold elevation in the level of carboxypeptidase Y (CPY) results when a multiple-copy plasmid containing the PRC1 gene is introduced into yeast. Unlike the situation with a single copy of PRC1 in which newly synthesized CPY is efficiently localized to the vacuole, plasmid-directed overproduction results in secretion of greater than 50% of the protein as the precursor form. Secretion is blocked in a mutant that is defective at a late stage in the transport of periplasmic proteins. Unlike normal cell surface glycoproteins, secreted CPY precursor acquires no additional oligosaccharide modifications beyond those that accompany normal transport to the vacuole. In the periplasm, the CPY precursor is proteolytically activated to an enzymatically active form by an enzyme that is unrelated to the vacuolar processing enzyme. These findings suggest that proper sorting and transport of CPY is saturable. This may reflect limiting amounts of a CPY-sorting receptor, or of CPY-modifying machinery that is essential for recognition by such a receptor.

Carboxypeptidases↗
PubMed1986

GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae.

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.

Biological Transport↗
PubMed1985

A test of clathrin function in protein secretion and cell growth.

Clathrin-coated membranes are intimately associated with a variety of protein transport processes in eukaryotic cells, yet no direct test of clathrin function has been possible. The data presented demonstrate that Saccharomyces cerevisiae does not require clathrin for either cell growth or protein secretion. Antiserum to the yeast clathrin heavy chain has been used to isolate a molecular clone of the heavy chain gene (CHC1) from a library of yeast DNA in lambda gt11. Clathrin-deficient mutant yeast have been obtained by replacing the single chromosomal CHC1 gene with a disrupted version of the cloned DNA. Cells harboring a nonfunctional chc1 allele produce no immunoreactive heavy chain polypeptide, and vesicles prepared from mutant cells are devoid of clathrin heavy and light chains. Although clathrin-deficient cells grow two to three times more slowly than normal, secretion of invertase occurs at a nearly normal rate. Therefore protein transport through the secretory pathway is not obligately coupled to the formation of clathrin-coated vesicles.

Biological Transport↗
PubMed1985

Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme.

The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme. Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2. Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added. Subsequent transport to the cell surface is not similarly delayed. One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein. Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage. While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue. s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme. This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body.

Biological Transport↗
PubMed1985

Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum.

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event.

Amino Acid Sequence↗
PubMed1984

Early steps in processing of yeast glycoproteins.

N-linked oligosaccharides have been examined on glycoproteins accumulated in yeast mutants that are blocked at successive stages in the secretory pathway, and in a new mutant, gls1-1, deficient in removal of glucose from N-linked core oligosaccharides, but not blocked in secretion. Oligosaccharides on invertase, a secreted protein, and carboxypeptidase Y, a vacuolar protein, are matured normally in the gls1 mutant but retain three glucoses/carbohydrate chain. The gls1 mutation is recessive and extracts of mutant cells are inactive in release of labeled glucose from core oligosaccharides. The mutant thus lacks glucosidase I activity but could also be deficient in the other core oligosaccharide glucosidase. When transport from the endoplasmic reticulum is blocked in sec18, N-linked oligosaccharides accumulate with a size corresponding to Man8GlcNAc2 when the normal GLS1 allele is present, and Glc3Man8GlcNAc2 in the gls1 mutant. From this we infer that all glucose units are removed prior to glycoprotein transport from the endoplasmic reticulum.

Carboxypeptidases↗
PubMed1984

Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway.

Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway. In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro. At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates. In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed. The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus. Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).

Genotype↗
PubMed1984

Isolation of glucose-containing high-mannose glycoprotein core oligosaccharides.

The total cell wall mannoprotein has been isolated from a mutant of Saccharomyces cerevisiae that fails to remove the glucose units of the dolichol-linked precursor after transfer of the oligosaccharide to asparagine units in the protein. The oligosaccharides released from this mannoprotein by endoglucosaminidase H digestion show 1H NMR signals assignable to three alpha-linked glucose units as delta 5.52, 5.27, and 5.17, and a comparison with the chemical shifts of reference compounds shows that these signals are consistent with the structure alpha Glc----2 alpha Glc----3 alpha Man----2. This provides a direct confirmation for the structure previously assigned to the lipid-linked precursor. Analysis of the larger oligosaccharides confirms that the presence of the glucose units does not prevent elongation of the alpha 1----6-linked polymannose backbone or addition of alpha 1----3-linked mannose to the core.

Glucose↗
PubMed1984

Yeast secretory mutants that block the formation of active cell surface enzymes.

Yeast cells secrete a variety of glycosylated proteins. At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth. Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C). sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum. Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of [3H]mannose into glycoprotein is reduced. Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein. In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C.

Acid Phosphatase↗
PubMed1984

Genes required for completion of import of proteins into the endoplasmic reticulum in yeast.

Yeast secretory mutants sec53 and sec59 define a posttranslational stage in the penetration of glycoprotein precursors into the endoplasmic reticulum (ER). In the previous report we showed that at the restrictive temperature (37 degrees C) these mutants accumulate enzymatically inactive and incompletely glycosylated forms of the secretory enzyme invertase and the vacuolar enzyme carboxypeptidase Y. Cell fractionation experiments reveal that these precursor forms remain firmly bound to the ER membrane. However, upon return to the permissive temperature (24 degrees C), the invertase precursors are glycosylated, become partially active, and are secreted. Thermoreversible conversion does not require protein synthesis, but does require energy. In contrast to the effect of these mutations, inhibition of oligosaccharide synthesis with tunicamycin at 37 degrees C causes irreversible accumulation of unglycosylated invertase. The effect of the drug is exaggerated by high temperature since unglycosylated invertase synthesized in the presence of tunicamycin at 25 degrees C is secreted. A portion of the invertase polypeptide accumulated at 37 degrees C is preserved when membranes from sec53 and sec59 are treated with trypsin. In the presence of Triton X-100 or saponin, the invertase is degraded completely. The protected fragment appears to represent a portion of the invertase polypeptide that is embedded in or firmly associated with the ER membrane. This association may develop early during the synthesis of invertase, so that in the absence of translocation, some of the completed polypeptide chain remains exposed on the cytoplasmic surface of the ER.

Biological Transport↗