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R Schekman

Publications and source records attributed to R Schekman.

At least 19 recordsLinked to original sources
PubMed2000

The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae.

To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S. cerevisiae (p24Delta8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24Delta8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single Deltaemp24 deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.

Base Sequence↗
PubMed2000

Pho86p, an endoplasmic reticulum (ER) resident protein in Saccharomyces cerevisiae, is required for ER exit of the high-affinity phosphate transporter Pho84p.

In the budding yeast Saccharomyces cerevisiae, PHO84 and PHO86 are among the genes that are most highly induced in response to phosphate starvation. They are essential for growth when phosphate is limiting, and they function in the high-affinity phosphate uptake system. PHO84 encodes a high-affinity phosphate transporter, and mutations in PHO86 cause many of the same phenotypes as mutations in PHO84, including a phosphate uptake defect and constitutive expression of the secreted acid phosphatase, Pho5p. Here, we show that the subcellular localization of Pho84p is regulated in response to extracellular phosphate levels; it is localized to the plasma membrane in low-phosphate medium but quickly endocytosed and transported to the vacuole upon addition of phosphate to the medium. Moreover, Pho84p is localized to the endoplasmic reticulum (ER) and fails to be targeted to the plasma membrane in the absence of Pho86p. Utilizing an in vitro vesicle budding assay, we demonstrate that Pho86p is required for packaging of Pho84p into COPII vesicles. Pho86p is an ER resident protein, which itself is not transported out of the ER. Interestingly, the requirement of Pho86p for ER exit is specific to Pho84p, because other members of the hexose transporter family to which Pho84 belongs are not mislocalized in the absence of Pho86p.

Adaptor Proteins, Vesicular Transport↗
PubMed2000

The use of liposomes to study COPII- and COPI-coated vesicle formation and membrane protein sorting.

We have established systems that reconstitute the biogenesis of coated transport vesicles with liposomes made of pure lipids and purified coat proteins. Optimization of the lipid composition in the liposomes allowed the efficient binding of both coat protein I and coat protein II (COPII) coat subunits. Coated vesicles of approximately the size generated from biomembranes were detected and characterized by centrifugation analysis and electron microscopy. A variation of this budding reaction allowed us to measure the sorting of v-SNARE proteins into synthetic COPII vesicles. We developed a novel system to tether glutathione S-transferase (GST)-hybrid proteins to the surface of liposomes formulated with a glutathione-derivatized phospholipid. This system allowed us to detect the positive role of cytoplasmic domains of two v-SNARE proteins that are packaged into COPII vesicles. Therefore, both generation of coated vesicles and protein sorting into the vesicles can be reproduced with liposomes and purified proteins.

Biological Transport↗
PubMed2000

Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae.

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.

Amino Acid Sequence↗
PubMed1999

Out of the ER--outfitters, escorts and guides.

The endoplasmic reticulum (ER) contains a variety of specialized proteins that interact with secretory proteins and facilitate their uptake into transport vesicles destined for the Golgi apparatus. These accessory proteins might induce and/or stabilize a conformation that is required for secretion competence or they might be directly involved in the sorting and uptake of secretory proteins into Golgi-bound vesicles. Recent efforts have aimed to identify and characterize the role of several of these substrate-specific accessory proteins.

Animals↗
PubMed1999

The engagement of Sec61p in the ER dislocation process.

Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm. We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S. cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER. Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells. Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation. We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes.

Alleles↗
PubMed1998

Reconstitution of retrograde transport from the Golgi to the ER in vitro.

Retrograde transport from the Golgi to the ER is an essential process. Resident ER proteins that escape the ER and proteins that cycle between the Golgi and the ER must be retrieved. The interdependence of anterograde and retrograde vesicle trafficking makes the dissection of both processes difficult in vivo. We have developed an in vitro system that measures the retrieval of a soluble reporter protein, the precursor of the yeast pheromone alpha-factor fused to a retrieval signal (HDEL) at its COOH terminus (Dean, N., and H.R.B Pelham. 1990. J. Cell Biol. 111:369-377). Retrieval depends on the HDEL sequence; the alpha-factor precursor, naturally lacking this sequence, is not retrieved. A full cycle of anterograde and retrograde transport requires a simple set of purified cytosolic proteins, including Sec18p, the Lma1p complex, Uso1p, coatomer, and Arf1p. Among the membrane-bound v-SNAP receptor (v-SNARE) proteins, Bos1p is required only for forward transport, Sec22p only for retrograde trafficking, and Bet1p is implicated in both avenues of transport. Putative retrograde carriers (COPI vesicles) generated from Golgi-enriched membranes contain v-SNAREs as well as Emp47p as cargo.

ADP-Ribosylation Factor 1↗
PubMed1998

Coatomer, Arf1p, and nucleotide are required to bud coat protein complex I-coated vesicles from large synthetic liposomes.

Synthetic coat protein complex I (COPI)-coated vesicles form spontaneously from large ( approximately 300 nm in diameter), chemically defined liposomes incubated with coatomer, Arf1p, and guanosine 5'-[gamma-thio]triphosphate. Coated vesicles are 40-70 nm in diameter, approximately the size of COPI vesicles formed from native membranes. The formation of COPI-coated buds and vesicles and the binding of Arf1p to donor liposomes depends on guanosine 5'-[gamma-thio]triphosphate. In contrast to the behavior of the COPII coat, coatomer binds to liposomes containing a variety of charged or neutral phospholipids. However, the formation of COPI buds and vesicles is stimulated by acidic phospholipids. In the absence of Arf1p, coatomer binds to liposomes containing dioleoylphosphatidic acid as a sole acidic phospholipid to form large coated surfaces without forming COPI-coated buds or vesicles. We conclude that Arf1p-GTP and coatomer comprise the minimum apparatus necessary to create a COPI-coated vesicle.

ADP-Ribosylation Factor 1↗
PubMed1998

Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi vesicle SNAREs.

Protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus involves specific uptake into coat protein complex II (COPII)-coated vesicles of secretory and of vesicle targeting (v-SNARE) proteins. Here, two ER to Golgi v-SNAREs, Bet1p and Bos1p, were shown to interact specifically with Sar1p, Sec23p, and Sec24p, components of the COPII coat, in a guanine nucleotide-dependent fashion. Other v-SNAREs, Sec22p and Ykt6p, might interact more weakly with the COPII coat or interact indirectly by binding to Bet1p or Bos1p. The data suggest that transmembrane proteins can be taken up into COPII vesicles by direct interactions with the coat proteins and may play a structural role in the assembly of the COPII coat complex.

Binding Sites↗
PubMed1998

COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.

COPII vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated vesicles.

COP-Coated Vesicles↗
PubMed1998

epsilon-COP is a structural component of coatomer that functions to stabilize alpha-COP.

We isolated a novel yeast alpha-COP mutant, ret1-3, in which alpha-COP is degraded after cells are shifted to a restrictive temperature. ret1-3 cells cease growth at 28 degrees C and accumulate the ER precursor of carboxypeptidase Y (p1 CPY). In a screen for high copy suppressors of these defects, we isolated the previously unidentified yeast epsilon-COP gene. epsilon-COP (Sec28p) overproduction suppresses the defects of ret1-3 cells up to 34 degrees C, through stabilizing levels of alpha-COP. Surprisingly, cells lacking epsilon-COP (sec28 Delta) grow well up to 34 degrees C and display normal trafficking of carboxypeptidase Y and KKXX-tagged proteins at a permissive temperature. epsilon-COP is thus non-essential for yeast cell growth, but sec28 Delta cells are thermosensitive. In sec28 Delta cells shifted to 37 degrees C, wild-type alpha-COP (Ret1p) levels diminish rapidly and cells accumulate p1 CPY; these defects can be suppressed by alpha-COP overproduction. Mutant coatomer from sec28 Delta cells behaves as an unusually large protein complex in gel filtration experiments. The sec28 Delta mutation displays allele-specific synthetic-lethal interactions with alpha-COP mutations: sec28 Delta ret1-3 double mutants are unviable at all temperatures, whereas sec28 Delta ret1-1 double mutants grow well up to 30 degrees C. Our results suggest that a function of epsilon-COP is to stabilize alpha-COP and the coatomer complex.

Alleles↗
PubMed1998

COPII-cargo interactions direct protein sorting into ER-derived transport vesicles.

Vesicles coated with coat protein complex II (COPII) selectively transport molecules (cargo) and vesicle fusion proteins from the endoplasmic reticulum (ER) to the Golgi complex. We have investigated the role of coat proteins in cargo selection and recruitment. We isolated integral membrane and soluble cargo proteins destined for transport from the ER in complexes formed in the presence of Sar1 and Sec23/24, a subset of the COPII components, and GTP or GMP-PNP. Vesicle fusion proteins of the vSNARE family and Emp24, a member of a putative cargo carrier family, were also found in COPII complexes. The inclusion of amino-acid permease molecules into the complex depended on the presence of Shr3, a protein required for the permease to leave the ER. Resident ER proteins Sec61, BiP (Kar2) and Shr3 were not included in the complexes, indicating that the COPII components bound specifically to vesicle cargo. COPII-cargo complexes and putative cargo adaptor-cargo complexes were also isolated from COPII vesicles. Our results indicate that cargo packaging signals and soluble cargo adaptors are recognized by a recruitment complex comprising Sar1-GTP and Sec23/24.

Amino Acid Transport Systems↗
PubMed1998

Coat assembly directs v-SNARE concentration into synthetic COPII vesicles.

COPII proteins are required to create transport vesicles and to select cargo molecules for transit from the ER. A reconstituted liposome budding reaction was used to detect the capture and concentration of membrane-associated v-SNARE molecules into synthetic COPII vesicles. A novel glutathione-phosphatidyl-ethanolamine conjugate (Glut-PE) was synthesized and incorporated into chemically defined liposomes to provide binding sites for GST hybrid proteins. Large liposomes containing bound cytoplasmic domains of the v-SNAREs, Sec22p or Bos1p, or of the ER resident proteins, Sec12p and Ufe1p, were exposed to COPII proteins and GMP-PNP. v-SNAREs but not resident proteins were concentrated in synthetic COPII vesicles generated from donor liposomes. We conclude that COPII proteins are necessary and sufficient for cargo selection and vesicle morphogenesis.

Biological Transport↗
PubMed1998

Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane.

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

Adenosine Triphosphate↗
PubMed1998

Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae.

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Delta strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4-1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.

Biological Transport, Active↗