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P J Novick

Publications and source records attributed to P J Novick.

At least 19 recordsLinked to original sources
PubMed2001

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors.

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.

Actins↗
PubMed2000

Ordering the final events in yeast exocytosis.

In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

Adenosine Triphosphatases↗
PubMed2000

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion.

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.

Cell Membrane↗
PubMed2000

The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles.

Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.

Actins↗
PubMed2000

A snc1 endocytosis mutant: phenotypic analysis and suppression by overproduction of dihydrosphingosine phosphate lyase.

The v-SNARE proteins Snc1p and Snc2p are required for fusion of secretory vesicles with the plasma membrane in yeast. Mutation of a methionine-based sorting signal in the cytoplasmic domain of either Sncp inhibits Sncp endocytosis and prevents recycling of Sncp to the Golgi after exocytosis. snc1-M43A mutant yeast have reduced growth and secretion rates and accumulate post-Golgi secretory vesicles and fragmented vacuoles. However, cells continue to grow and secrete for several hours after de novo Snc2-M42A synthesis is repressed. DPL1, the structural gene for dihydrosphingosine phosphate lyase, was selected as a high copy number snc1-M43A suppressor. Because DPL1 also partially suppresses the growth and secretion phenotypes of a snc deletion, we propose that enhanced degradation of dihydrosphingosine-1-phosphate allows an alternative protein to replace Sncp as the secretory vesicle v-SNARE.

Aldehyde-Lyases↗
PubMed2000

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae.

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.

Actin Cytoskeleton↗
PubMed1999

Sec1p binds to SNARE complexes and concentrates at sites of secretion.

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

Adenosine Triphosphatases↗
PubMed1999

Promiscuity in Rab-SNARE interactions.

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.

Exocytosis↗
PubMed1999

The tail of a yeast class V myosin, myo2p, functions as a localization domain.

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant-negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2-66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 x g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.

Actins↗
PubMed1998

Identification of a Sec4p GTPase-activating protein (GAP) as a novel member of a Rab GAP family.

A yeast open reading frame sharing homology with the two known yeast Rab GTPase-activating proteins (GAPs), Gyp6p and Gyp7p, was found in a data base search. We have named the gene containing this open reading frame GYP1. Recombinant Gyp1p showed GAP activity on Sec4p, increasing both its steady-state rate and single turnover GTPase activity. Gyp1p also stimulated the GTPase activity of several other yeast Rab proteins including Ypt1p, Ypt7p, and Ypt51p but showed no GAP activity on Ypt6p and Ypt32p. Deletion of the GYP1 gene or overexpression of Gyp1p did not alter the growth rate of yeast. However, overexpression of Gyp1p was inhibitory in combination with a subset of secretory mutants including sec4-8 and several ypt1 mutants. This effect is probably due to the increase in GAP activity, which can be observed in a lysate from cells overexpressing Gyp1p. The finding that yeast Rab GAPs share homology with proteins in other species, such as Caenorhabditis elegans and human, suggests the existence of a conserved Rab GAP family.

Amino Acid Sequence↗
PubMed1997

Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles.

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.

Amino Acid Sequence↗
PubMed1994

GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway.

GTP binding proteins of the Sec4/Ypt/rab family regulate distinct vesicular traffic events in eukaryotic cells. We have cloned GDI1, an essential homolog of bovine rab GDI (GDP dissociation inhibitor) from the yeast Saccharomyces cerevisiae. Analogous to the bovine protein, purified Gdi1p slows the dissociation of GDP from Sec4p and releases the GDP-bound form from yeast membranes. Depletion of Gdi1p in vivo leads to loss of the soluble pool of Sec4p and inhibition of protein transport at multiple stages of the secretory pathway. Complementation analysis indicates that GDI1 is allelic to sec19-1. These results establish that Gdi1p plays an essential function in membrane traffic and are consistent with a role for Gdi1p in the recycling of proteins of the Sec4/Ypt/rab family from their target membranes back to their vesicular pools.

Amino Acid Sequence↗
PubMed1993

Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae. Conservation of GDI membrane dissociation activity.

Rab GDP dissociation inhibitor (Rab GDI), will induce the dissociation of GDP-bound rab3A from synaptic membranes and will inhibit GDP dissociation from Sec4, a member of the Rab subgroup of the Ras GTPase superfamily which is required for exocytosis in Saccharomyces cerevisiae. We report that Rab GDI releases GDP-bound Sec4 from yeast membranes. dGDI, a Drosophila homologue can similarly inhibit GDP dissociation from Sec4 and release GDP-bound Sec4 from yeast membranes. An activity partially purified from yeast cytosol dissociates GDP-bound Sec4 from yeast membranes, suggesting that yeast also possess a GDI protein that functions to recycle Sec4 from its target membrane.

Animals↗
PubMed1991

A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.

Animals↗
PubMed1990

Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic.

The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.

Binding, Competitive↗