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Biomedical subjects

R Schekman

Publications and source records attributed to R Schekman.

At least 19 recordsLinked to original sources
PubMed1996

Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles.

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaflet clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.

COP-Coated Vesicles↗
PubMed1996

Selective uptake of cytosolic, peroxisomal, and plasma membrane proteins into the yeast lysosome for degradation.

When glucose-starved cells are replenished with glucose, the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the yeast lysosome (vacuole) for degradation. The glucose-induced targeting of FBPase to the vacuole for degradation occurs in cells grown under a variety of metabolic conditions. Immunoelectron microscopic studies demonstrate that the uptake of FBPase by the vacuole is mediated in part by an autophagic process. FBPase can be found on the vacuolar membrane and also at the sites of membrane invaginations. Furthermore, FBPase is associated with different forms of vesicles, which are induced to accumulate inside the vacuole. We have identified peroxisomes as the organelles that are delivered to the vacuole for degradation when cells are replenished with glucose. Ultrastructural studies indicate that peroxisomes are engulfed by the vacuole by an autophagic process, leading to the destruction of whole organelles in the vacuole. Furthermore, the galactose transporter (Gal2p) is also delivered from the plasma membrane to the vacuole for degradation in response to glucose. Gal2p is delivered to the vacuole through the endocytic pathway, as mutants defective in receptor-mediated endocytosis fail to degrade Gal2p in response to glucose.

Aspartic Acid Endopeptidases↗
PubMed1996

Coat proteins and vesicle budding.

The trafficking of proteins within eukaryotic cells is achieved by the capture of cargo and targeting molecules into vesicles that bud from a donor membrane and deliver their contents to a receiving department. This process is bidirectional and may involve multiple organelles within a cell. Distinct coat proteins mediate each budding event, serving both to shape the transport vesicle and to select by direct or indirect interaction the desired set of cargo molecules. Secretion, which has been viewed as a default pathway, may require sorting and packaging signals on transported molecules to ensure their rapid delivery to the cell surface.

Biological Transport↗
PubMed1996

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.

Biological Transport↗
PubMed1996

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

ATP-Binding Cassette Transporters↗
PubMed1996

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

Animals↗
PubMed1996

New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex.

We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of alpha-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the alpha subunit of coatomer.

Biological Transport↗
PubMed1995

COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast.

The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sar1p generate protein transport vesicles by forming the membrane coat termed COPII. We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope. Formation of COPI-coated, but not COPII-coated, buds and vesicles on the nuclear envelope is inhibited by the fungal metabolite brefeldin A. The two vesicle populations are distinct. However, both vesicle types are devoid of endoplasmic reticulum (ER) resident proteins, and each contains targeting proteins necessary for docking at the Golgi complex. Our data suggest that COPI and COPII mediate separate vesicular transport pathways from the ER.

Anti-Bacterial Agents↗
PubMed1995

Uncoupled packaging of targeting and cargo molecules during transport vesicle budding from the endoplasmic reticulum.

Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Golgi apparatus involves a mechanism that sorts and packages two classes of molecules into transport vesicles: targeting molecules, which are required for targeting and consumption of vesicular intermediates, and cargo proteins. In order to examine the importance of cargo in this packaging reaction, we developed an in vitro assay that quantifies vesicle formation based on segregation of targeting molecules. Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of targeting molecule-containing vesicles in a fashion indistinguishable from its normal counterpart. This observation implies that packaging of cargo proteins may be uncoupled from the recruitment of targeting molecules during vesicle budding from the endoplasmic reticulum. Using the same assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p).

COP-Coated Vesicles↗
PubMed1995

BiP and Sec63p are required for both co- and posttranslational protein translocation into the yeast endoplasmic reticulum.

Two interacting heat shock cognate proteins in the lumen of the yeast endoplasmic reticulum (ER), Sec63p and BiP (Kar2p), are required for posttranslational translocation of yeast alpha-factor precursor in vitro. To investigate the role of these proteins in cotranslational translocation, we examined the import of invertase into wild-type, sec63, and kar2 mutant yeast membranes. We found that Sec63p and Kar2p are necessary for both co- and posttranslational translocation in yeast. Several kar2 mutants, one of which had normal ATPase activity, were defective in cotranslational translocation of invertase. We conclude that the requirement for BiP/Kar2p, which is not seen in a reaction reconstituted with pure mammalian membrane proteins [Görlich, D. & Rapoport, T.A. (1993) Cell 75, 615-630], is not due to a distinction between cotranslational translocation in mammalian cells and posttranslational translocation in yeast cells.

Biological Transport↗
PubMed1995

Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes.

The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment. We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events.

Adenosine Triphosphatases↗
PubMed1995

Interaction between BiP and Sec63p is required for the completion of protein translocation into the ER of Saccharomyces cerevisiae.

To clarify the roles of Kar2p (BiP) and Sec63p in translocation across the ER membrane in Saccharomyces cerevisiae, we have utilized mutant alleles of the essential genes that encode these proteins: kar2-203 and sec63-1. Sanders et al. (Sanders, S. L., K. M. Whitfield, J. P. Vogel, M. D. Rose, and R. W. Schekman. 1992. Cell. 69:353-365) showed that the translocation defect of the kar2-203 mutant lies in the inability of the precursor protein to complete its transit across the membrane, suggesting that the lumenal hsp70 homologue Kar2p (BiP) binds the transiting polypeptide in order to facilitate its passage through the pore. We now show that mutation of a conserved residue (A181-->T) (Nelson, M. K., T. Kurihara, and P. Silver. 1993. Genetics. 134:159-173) in the lumenal DnaJ box of Sec63p (sec63-1) results in an in vitro phenotype that mimics the precursor stalling defect of kar2-203. We demonstrate by several criteria that this phenotype results specifically from a defect in the lumenal interaction between Sec63p and BiP: Neither a sec62-1 mutant nor a mutation in the cytosolically exposed domain of Sec63p causes precursor stalling, and interaction of the sec63-1 mutant with the membranebound components of the translocation apparatus is unimpaired. Additionally, dominant KAR2 suppressors of sec63-1 partially relieve the stalling defect. Thus, proper interaction between BiP and Sec63p is necessary to allow the precursor polypeptide to complete its transit across the membrane.

Binding Sites↗
PubMed1994

Coatomer-rich endoplasmic reticulum.

We identify in normal cells the existence of two distinct sites of the transitional endoplasmic reticulum (ER), one housing the Sec23p protein complex (the classical transitional element), the other the coatomer protein complex (the coatomer-rich ER). Experimental conditions that reduce transport from the ER to the Golgi complex lead to the overexpression of this newly defined coatomer-rich ER.

Animals↗