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P Novick

Publications and source records attributed to P Novick.

At least 37 records · Page 2Linked to original sources
PubMed1997

Interactions of nucleotide release factor Dss4p with Sec4p in the post-Golgi secretory pathway of yeast.

SEC4 is an essential gene encoding a small GTPase that is involved in Golgi to cell surface transport in Saccharomyces cerevisiae and is a paradigm for studies on the mode of action of Rab proteins. We describe here the features of interaction of Sec4p with the accessory protein Dss4p. Dss4p is found both on membranes and in the cytosol; however, it is the membrane fraction that is complexed to Sec4p. Dss4p, like its mammalian counterpart, Mss4, binds zinc, and disruption of the zinc-binding site disrupts the ability of the protein to interact with Sec4p. DSS4 overexpression can rescue the lethal phenotype of two alleles of SEC4, corresponding to dominant mutations of Ras. We demonstrate that this suppression is due to the ability of Dss4p to form a tight complex with the mutant forms of Sec4p and hence sequester the mutant protein from its inhibitory effect. These results imply an in vivo role for Dss4p as a guanine nucleotide dissociation stimulator. In vitro the protein has the ability to stimulate the dissociation rate of both GDP and GTP from Sec4p. We examined the relationship of GDI1 and DSS4 with SEC4 both genetically and biochemically. These results exclude a role for DSS4 in the recruitment of Sec4p/GDI onto membranes.

Alleles↗
PubMed1997

Identification and characterization of homologues of the Exocyst component Sec10p.

The SEC10 gene product is a member of the Exocyst complex essential for exocytosis in the budding yeast Saccharomyces cerevisiae. We report here the cloning and characterization of human Sec10p (hSec10p; GenBank accession number U85946). hSec10p is a 77-kDa protein with 23% amino acid identity to yeast Sec10p and 37% identity to a C. elegans protein found in the database. Northern and Western blot analyses indicate that hSec10 has a broad tissue distribution. Immunofluorescence staining of COS cells cotransfected with hSec10p and a mammalian Sec8p demonstrates that these two proteins have an identical distribution in the cell including a localization in the peripheral cytoplasm. These data suggest that hSec10p is a component of the mammalian counterpart of the yeast Exocyst complex essential for post-Golgi traffic.

Amino Acid Sequence↗
PubMed1997

The diversity of Rab proteins in vesicle transport.

Rab proteins have been primarily implicated in vesicle docking as regulators of SNARE pairing. Recent findings, however, indicate that their function in vesicle trafficking can go beyond this role, and a number of proteins, unrelated to each other, have been identified as putative Rab effectors. Although the GTPase switch of Rab proteins is highly conserved, functional mechanisms may be highly diversified among members of the Rab family.

Amino Acid Sequence↗
PubMed1997

Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.

Actins↗
PubMed1996

The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S. cerevisiae. Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6-Sec8-Sec15 complex, indicating that these genes may encode some of the remaining components of this complex. To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co-immunoprecipitation studies. Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6-Sec8-Sec15 complex for peptide microsequencing. The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components. In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70. Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst.

Amino Acid Sequence↗
PubMed1996

Phosphoinositides as regulators in membrane traffic.

Phosphorylated products of phosphatidylinositol play critical roles in the regulation of membrane traffic, in addition to their classical roles as second messengers in signal transduction at the cell surface. Growing evidence suggests that phosphorylation-dephosphorylation of the polar heads of phosphoinositides (polyphosphorylated inositol lipids) in specific intracellular locations signals either the recruitment or the activation of proteins essential for vesicular transport. Cross talk between phosphatidylinositol metabolites and guanosine triphosphatases is an important feature of these regulatory mechanisms.

Animals↗
PubMed1995

The role of Myo2, a yeast class V myosin, in vesicular transport.

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post-Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy-peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.

Biological Transport↗
PubMed1995

Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis.

Amino Acid Sequence↗
PubMed1994

Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis.

To identify potential Sec4 effectors, we isolated high copy suppressors of a Sec4 effector domain mutant. The most potent of these was found to be SEC9, a gene required for post-Golgi transport. The sole essential domain of Sec9 has significant sequence similarity to the neuronal protein SNAP-25, a component of the SNARE complex, that is implicated in vesicle targeting and fusion. Analogous to SNAP-25, Sec9 is bound to the yeast plasma membrane and is absent from post-Golgi vesicles. Furthermore, Sec9 is physically associated with two proteins that are homologous to components of the neuronal SNARE complex. Our results identify Sec9 as the yeast cognate of SNAP-25 and suggest that SNARE complexes acting at specific stages of vesicular transport serve as the ultimate targets of regulation by members of the Sec4/Ypt1/Rab family of GTPases.

Amino Acid Sequence↗
PubMed1993

Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae.

The yeast S. cerevisiae possesses two genes, SNC1 and SNC2, that encode homologs of vertebrate synaptic vesicle-associated membrane proteins, also known as synaptobrevins. Here we describe the cloning of SNC2 and demonstrate that yeast lacking both SNC genes are deficient in normal bulk secretion, accumulate large numbers of post-Golgi vesicles, and display a variety of conditional lethal phenotypes. In addition, we show that yeast Snc proteins localize to post-Golgi transport vesicles that accumulate in a late-acting sec mutant. Our findings clearly place the Snc proteins on the late secretory pathway in S. cerevisiae and provide direct in vivo evidence that synaptobrevin-like proteins mediate the targeting and transport of secretory proteins.

Amino Acid Sequence↗
PubMed1993

Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4.

The genes SEC4 and YPT1 encode Ras-related GTP-binding proteins in the yeast Saccharomyces cerevisiae. Ypt1 is necessary for vesicular transport from the endoplasmic reticulum to the Golgi, whereas Sec4 is required for fusion of post-Golgi secretory vesicles to the plasma membrane. Recently, three structural domains have been proposed to specify the stage in cellular transport at which members of the Sec4/Ypt1/Rab family act: the effector domain, the C-terminal hypervariable region, and a region corresponding to loop 7 in the structure of p21ras (ref. 8). Here we use Sec4/Ypt1 chimaeras to show that these three regions cooperate to specify Ypt1 function and that the C-terminal hypervariable region is needed for Ypt1 localization to the Golgi. Unexpectedly, we found that a single chimaera can function as either Ypt1 or Sec4 without missorting carboxypeptidase Y or invertase.

Amino Acid Sequence↗
PubMed1993

DSS4-1 is a dominant suppressor of sec4-8 that encodes a nucleotide exchange protein that aids Sec4p function.

The protein Sec4p plays an essential role at the final stage of the yeast secretory pathway and belongs to the ras superfamily of GTP-binding proteins, more specifically to a branch that includes Ypt1p in Saccharomyces cerevisiae and rab proteins in mammalian cells. GTP-binding proteins change conformation depending on whether GTP or GDP is bound and can thus act as a regulatory switch. The protein remains in its inactive, GDP-bound form until exchange of GTP for GDP allows it to stimulate a downstream effector. This interaction is curtailed by GTP hydrolysis. The rates of nucleotide exchange and GTP hydrolysis can be regulated by interaction with accessory proteins. Although GDP dissociation stimulators (GDS) have been identified that act on members of the ras and rho branches of the superfamily, less is known regarding GDSs that act on members of the Sec4/Ypt1/Rab subgroup. A preliminary characterization of a Rab3A GDP dissociation stimulating activity has been presented. We report here the use of suppressor analysis to clone a gene, dss4, encoding a 17K protein that aids Sec4p action in vivo by functioning as a GDP dissociation stimulator.

Amino Acid Sequence↗
PubMed1993

A mammalian guanine-nucleotide-releasing protein enhances function of yeast secretory protein Sec4.

Small GTP-binding proteins of the ras superfamily are important for exocytosis from eukaryotic cells. GTP-binding proteins can exist in two different conformations depending on whether they are bound to GDP or GTP, and are thought to function as molecular switches that regulate a variety of cellular processes. The GTP-GDP cycle is controlled by accessory proteins that promote the exchange of bound GDP or the hydrolysis of GTP. The protein Sec4, a member of the Sec4/Ypt1/Rab branch of the Ras superfamily, is involved in a late stage of the secretory pathway in yeast. Here we report the isolation of a mammalian complementary DNA, mss4, encoding a GDP-releasing protein that enhances Sec4 function. The Mss4 protein also stimulates GDP release from Ypt1 and from the mammalian protein Rab3a, but not from Ras2. Mss4 shows sequence similarity to Dss4, a yeast protein with similar biochemical properties.

ATP-Binding Cassette Transporters↗