Biomedical subjects

J E Gerst

Publications and source records attributed to J E Gerst.

At least 19 recordsLinked to original sources

PubMed2001

t-SNARE dephosphorylation promotes SNARE assembly and exocytosis in yeast.

The role of protein phosphorylation in secretion is not well understood. Here we show that yeast lacking the Snc1,2 v-SNAREs, or bearing a temperature-sensitive mutation in the Sso2 t-SNARE, are rescued at restrictive conditions by the addition of ceramide precursors and analogs to the growth medium. Rescue results from dephosphorylation of the Sso t-SNAREs by a ceramide-activated type 2A protein phosphatase (Sit4) involved in cell cycle control. Sso t-SNARE dephosphorylation correlated with its assembly into complexes with the Sec9 t-SNARE, both in vitro and in vivo, and with an increase in protein trafficking and secretion in cells. SNARE complexes isolated under these conditions contained only Sso and Sec9, suggesting that a t-t-SNARE fusion complex is sufficient to confer exocytosis. Mutation of a single PKA site (Ser79 to Ala79) in Sso1 resulted in a decrease in phosphorylation and was sufficient to confer growth to snc cells at restrictive conditions. Thus, modulation of t-SNARE phosphorylation regulates SNARE complex assembly and membrane fusion in vivo.

Exocytosis↗

PubMed2000

Yeast exocytic v-SNAREs confer endocytosis.

In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1(ala43)), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1(ala43) yeast are deficient in their ability to deliver the soluble dye FM4-64 to the vacuole. Under conditions in which vesicles accumulate, FM4-64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, alpha-factor-stimulated endocytosis of the alpha-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1(ala43) was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane.

Base Sequence↗

PubMed1999

SNAREs and SNARE regulators in membrane fusion and exocytosis.

Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles. This apparatus comprises the secretory (or 'protein export') pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles. Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another. Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion. These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e. the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion. SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events. In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g. 'SNARE-masters') in mediating events leading to membrane fusion. A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented.

Animals↗

PubMed1999

Yeast VSM1 encodes a v-SNARE binding protein that may act as a negative regulator of constitutive exocytosis.

We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomyces cerevisiae. A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell extracts. Disruption of the VSM1 gene results in an increase of proteins secreted into the medium but does not affect the processing or secretion of invertase. In contrast, VSM1 overexpression in cells which bear a temperature-sensitive mutation in the Sec9 t-SNARE (sec9-4 cells) results in the accumulation of non-invertase-containing low-density secretory vesicles, inhibits cell growth and the secretion of proteins into the medium, and blocks rescue of the temperature-sensitive phenotype by SNC1 overexpression. Yet, VSM1 overexpression does not affect yeast bearing a sec9-7 allele which, in contrast to sec9-4, encodes a t-SNARE protein capable of forming a stable SNARE complex in vitro at restrictive temperatures. On the basis of these results, we propose that Vsm1 is a novel v-SNARE-interacting protein that appears to act as negative regulator of constitutive exocytosis. Moreover, this regulation appears specific to one of two parallel exocytic paths which are operant in yeast cells.

Amino Acid Sequence↗

PubMed1998

Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast.

Members of the synaptobrevin/VAMP family of v-SNAREs are thought to be essential for vesicle docking and exocytosis in both lower and higher eukaryotes. Here, we describe yeast mutants that appear to bypass the known v-SNARE requirement in secretion. Recessive mutations in either VBM1 or VBM2, which encode related ER-localized membrane proteins, allow yeast to grow normally and secrete in the absence of Snc v-SNAREs. These mutants show selective alterations in protein transport, resulting in the differential trafficking and secretion of certain protein cargo. Yet, processing of the vacuolar marker, carboxypeptidase Y, and the secreted protein, invertase, appear normal in these mutants indicating that general protein trafficking early in the pathway is unaffected. Interestingly, VBM1 and VBM2 are allelic to ELO3 and ELO2, two genes that have been shown recently to mediate the elongation of very long chain fatty acids and subsequent ceramide and inositol sphingolipid synthesis. Thus, the v-SNARE requirement in constitutive exocytosis is abrogated by mutations in early components of the secretory pathway that act at the level of lipid synthesis to affect the ability of secretory vesicles to sort and deliver protein cargo.

Alleles↗

PubMed1997

Conserved alpha-helical segments on yeast homologs of the synaptobrevin/VAMP family of v-SNAREs mediate exocytic function.

We are studying yeast homologs of the synaptobrevin/VAMP family of vesicle-associated membrane proteins, which act as vesicular compartment-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) in cells having a capacity for stimulus-coupled secretion, as well as in other cell types. The yeast homologs, Snc1 and Snc2, localize to secretory vesicles and are required for normal bulk secretion in Saccharomyces cerevisiae. Here we have used Snc deletion mutants and chimeric Snc-VAMP proteins to demonstrate that these v-SNAREs can be dissected into regions that are either indispensable or dispensable for exocytic function in vivo. We have found that a region encompassing two predicted amphipathic alpha-helices (helix 1 and helix 2) (residues 32-85), which are thought to form coiled-coil structures, is essential for conferring exocytosis in yeast. Deletions in either the helix 1 or helix 2 segments result in a complete loss in the ability of the protein to confer secretion competence to snc cells and to interact genetically with components of the proposed fusion complex: the Sec9 and Sso2 t-SNAREs and the Sec17 alpha-SNAP homolog. In contrast, deletions in either the variable (residues 2-27) or putative intravesicular (residues 115-117) regions have no deleterious effect upon v-SNARE function. This makes it unlikely that sequences in either the amino or carboxyl terminus act in an exocytic capacity. Along with additional studies utilizing chimeric Snc-VAMP proteins, we suggest that although the Snc and synaptobrevin/VAMP proteins have evolved to mediate vastly different exocytic programs, their structural requirements and actions have remained remarkably well-conserved in evolution.

Base Sequence↗

PubMed1997

Identification of a novel Ca2+-dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in PLD1.

We have previously reported the identification and partial characterization of a gene encoding a phospholipase D activity (PLD1) in the yeast, Saccharomyces cerevisiae. Here we report the existence of a second phospholipase D activity, designated PLD2, in yeast cells bearing disruption at the PLD1 locus. PLD2 is a Ca2+-dependent enzyme which preferentially utilizes phosphatidylethanolamine over phosphatidylcholine as a substrate. In contrast to PLD1, the activity of PLD2 is insensitive to phosphatidylinositol 4,5-bisphosphate, and the enzyme is incapable of catalyzing the transphosphatidylation reaction with short chain alcohols as acceptors. Subcellular fractionation shows that PLD2 localizes mainly to the cytosol, but could also be detected in the particulate fraction. Thus, the biochemical properties of PLD2 appear to be substantially different from those of PLD1. PLD2 activity is significantly and transiently elevated upon exit of wild type yeast cells from stationary phase, suggesting that it may play a role in the initiation of mitotic cell division in yeast. In view of the significantly different properties of PLD1 and PLD2, and because the yeast genome contains PLD1 as the sole member of the recently defined PLD gene family, it may be concluded that PLD2 is structurally unrelated to PLD1. Thus, the novel PLD2 activity described herein is likely to represent the first identified member of a new PLD gene family.

Calcium↗

PubMed1996

Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs. Dimerization and actin binding.

The yeast adenylyl cyclase-associated protein, CAP, was identified as a component of the RAS-activated cyclase complex. CAP consists of two functional domains separated by a proline-rich region. One domain, which localizes to the amino terminus, mediates RAS signaling through adenylyl cyclase, while a domain at the carboxyl terminus is involved in the regulation of cell growth and morphogenesis. Recently, the carboxyl terminus of yeast CAP was shown to sequester actin, but whether this function has been conserved, and is the sole function of this domain, is unclear. Here, we demonstrate that the carboxyl-terminal domains of CAP and CAP homologs have two separate functions. We show that carboxyl-terminals of both yeast CAP and a mammalian CAP homolog, MCH1, bind to actin. We also show that this domain contains a signal for dimerization, allowing both CAP and MCH1 to form homodimers and heterodimers. The properties of actin binding and dimerization are mediated by separate regions on the carboxyl terminus; the last 27 amino acids of CAP being critical for actin binding. Finally, we present evidence that links a segment of the proline-rich region of CAP to its localization in yeast. Together, these results suggest that all three domains of CAP proteins are functional.

Actins↗

PubMed1996

Identification and characterization of a gene encoding phospholipase D activity in yeast.

We have identified an open reading frame on chromosome XI of the yeast, Saccharomyces cerevisiae, as encoding a protein with phospholipase D (PLD) activity. We have named this open reading frame, PLD1, and show that yeast bearing a disruption in this gene are unable to catalyze the hydrolysis of phosphatidylcholine. PLD1 encodes a hypothetical protein of 1683 amino acids and has a predicted molecular mass of 195 kDa. Yeast bearing disruptions at the PLD1 locus are morphologically normal and grow vegetatively like wild-type cells. In contrast, homozygous delta pld1 diploid cells are unable to sporulate and do not produce asci under conditions that induce meiosis and sporulation in wild-type cells. Thus, PLD1 is likely to be essential for the meiotic cycle in yeast cells. This is the first identification of a eukaryotic, nonplant, phosphatidylcholine-hydrolyzing phospholipase D gene. Because the biological role of PLD is not well understood, we expect that delta pld1 yeast will become a useful tool for the characterization of PLD functions as well as for the identification of mammalian PLD homologs.

Amino Acid Sequence↗

PubMed1995

Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.

Amino Acid Sequence↗

PubMed1994

Yeast Snc proteins complex with Sec9. Functional interactions between putative SNARE proteins.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated proteins that are proposed to be involved in membrane recognition and to act as receptors for components of the fusion machinery in neurons. We have previously described the yeast homologs, Snc1 and Snc2, and demonstrated that they localize to secretory vesicles and are required for normal secretion. Yeast lacking Snc protein expression accumulate post-Golgi transport vesicles that contain secretory proteins. Therefore, Snc proteins are essential for the fusion of carrier vesicles with the plasma membrane, and this property appears to have been conserved in evolution. We have now examined whether Snc proteins interact with other components of the late secretory pathway in yeast. Here we show that Snc proteins form a tight genetic and physical interaction with a plasma membrane protein, Sec9. Sec9 is the yeast equivalent of SNAP-25, a second receptor protein from neurons that has been shown to interact with synaptobrevin. We suggest, then, that recognition of the plasma membrane by secretory vesicles may involve the formation of a Snc-Sec9 complex and that this interaction has evolved as a fundamental step in secretory processes.

Cytoplasmic Granules↗

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

Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae.

We have isolated a rat cDNA whose expression suppresses the physiological consequences of the chromosomal disruption of CAP, the gene encoding the adenylyl cyclase-associated protein of Saccharomyces cerevisiae. Yeast CAP is a bifunctional protein: the NH2 terminus is necessary and sufficient for cellular responsiveness to activated RAS proteins, while the COOH terminus is required for normal cellular morphology and growth control. The rat MCH1 cDNA encodes a protein of 474 amino acids that is 36% identical to S. cerevisiae CAP and is capable of suppressing the loss of the COOH-terminal functions of CAP when expressed in yeast. The MCH1 protein therefore appears to be a structural and functional homolog of the yeast cyclase-associated proteins. Northern analysis of MCH1 gene expression shows it to be constitutively expressed in all cell and tissue types examined. The cloning of a rat homolog of CAP, in addition to the cloning of a human CAP homolog by Matviw et al. (Matviw, H., Yu, G., and Young, D. (1992) Mol. Cell. Biol. 12, 5033-5040), demonstrates that both cyclase-associated proteins and their functions may have evolved with mammalian cells.

Adaptor Proteins, Signal Transducing↗

PubMed1992

SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes.

SNC1, a gene from the yeast Saccharomyces cerevisiae, encodes a homolog of vertebrate synaptic vesicle-associated membrane proteins (VAMPs) or synaptobrevins. SNC1 was isolated by its ability to suppress the loss of CAP function in S. cerevisiae strains possessing an activated allele of RAS2. CAP is a component of the RAS-responsive S. cerevisiae adenylyl cyclase complex. The N-terminal domain of CAP is required for full cellular responsiveness to activated RAS proteins. The C-terminal domain of CAP is required for normal cellular morphology and responsiveness to nutrient extremes. Multicopy plasmids expressing SNC1 suppress only the loss of the C-terminal functions of CAP and only in the presence of activated RAS2.

Amino Acid Sequence↗

PubMed1991

CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex.

CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. We describe here a molecular and phenotypic analysis of the CAP protein. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.

Adenylyl Cyclases↗

PubMed1989

Phorbol ester impairs melanotropin receptor function and stimulates growth of cultured M2R melanoma cells.

We have examined the effects of a biologically active tumor promoting phorbol ester (phorbol 12-myristate, 13-acetate (PMA] which activates protein kinase C (PKC) on melanotropin receptor function and cell growth in the M2R mouse melanoma cell clone. Treatment of M2R cells with PMA resulted in a significant loss of beta-MSH binding. The effect was both time- and concentration-dependent. The inhibition of beta-MSH binding resulted from a decrease (greater than 85%) in active membranal receptors available on the external cell surface and not from either enhanced internalization or change in the binding affinity. Agonist-stimulated cyclic AMP accumulation was profoundly increased in a non-selective manner following short-term incubation (3 h) with PMA. This effect was completely reversed during long-term (72-96 h) incubation with the tumor promoting agent. Long-term culturing of M2R cells with PMA resulted in enhanced (+50%) proliferation of the melanoma cells. This enhancement was blocked by the addition of agents which stimulate the production of cAMP. Hence, phorbol esters are powerful growth promoters in transformed melanocytes and our findings indicate that the effects of melanotropins are selectively impaired during the process of growth promotion.

Animals↗

PubMed1988

A synthetic analog of the calmodulin-binding domain of myosin light chain kinase inhibits melanotropin receptor function and activation of adenylate cyclase.

In this study a synthetic analog of the calmodulin-binding domain of myosin light chain kinase, a 17-amino-acid peptide (M5) was used to examine the possible role of calmodulin in melanotropin receptor function. Binding of beta-melanocyte-stimulating hormone to its membrane receptor and subsequent stimulation of adenylate cyclase (AC) were found to be specifically inhibited by M5 in a dose-dependent and noncompetitive manner, both in intact M2R melanoma cells and in a plasma membrane preparation derived thereof. Half-maximal inhibition of both hormone binding and melanotropin-sensitive AC activity was shown to occur at approximately 1 microM M5. In contrast, stimulation of AC by prostaglandin E1, guanosine 5'-O-(3-thio)triphosphate, forskolin, and unstimulated enzyme activity were unaffected by the presence of M5, under the same assay conditions. Furthermore, addition of a molar excess of calmodulin to the AC assay completely abolished the inhibitory effects of the peptide on melanotropin-stimulated AC activity. Other peptides of similar size, which bind to calmodulin with low affinity (e.g. glucagon, somatostatin, and vasoactive intestinal peptide), were shown to be totally ineffective in inhibiting melanotropin-sensitive AC. These findings, along with those shown previously for other antagonists of calmodulin, suggest a role for an M5-binding protein, as of yet unidentified, involved in the regulation of the melanotropin receptor function.

Adenylyl Cyclases↗

PubMed1988

Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling.

In this study, two melanotropin binding proteins from M2R melanoma cells have been identified based on the photochemical cross-linking of [125I]iodinated porcine beta-MSH ([ 125I]iodo-beta-MSH) to melanoma cell membranes, using N-hydroxysuccinimidyl-azidobenzoate. Autoradiography of photoaffinity-labeled M2R membrane protein, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the specific labeling of two separate bands with an apparent molecular mass of 43 and 46 kilodaltons, respectively. Photoaffinity labeling of both bands was of near-equal intensity and could be inhibited, in a dose-dependent manner, by the addition of unlabeled beta-MSH before photolysis. In addition, agents known to inhibit the binding of beta-MSH to its cellular receptor, such as EGTA, GTP, guanosine 5'-O-(3-thio)triphosphate, and a synthetic analog of the calmodulin-binding domain of myosin light chain kinase-M5, were all found to specifically inhibit the labeling of these two protein bands by the azido derivative of [125I]iodo-beta-MSH. In contrast, addition of a nonrelated peptide, vasoactive intestinal peptide, had no effect upon the labeling of these melanotropin-binding proteins. On the basis of these results we suggest that the two proteins may function as the binding domain(s) of the cellular receptor for melanotropins, or may represent entire receptor moieties themselves.

Affinity Labels↗