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PubMed1992

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.

3T3 Cells↗

PubMed1992

Interactions of the ras-like protein p25rab3A with Mg2+ and guanine nucleotides.

The rab3A gene product is a 25 kDa guanine-nucleotide-binding protein which is expressed at high levels in neural tissue and has about 30% sequence similarity to ras. Purified p25rab3A has been used as substrate to examine its kinetics of nucleotide binding and hydrolysis, and to study the effects of Mg2+ on these processes. p25rab3A binds GDP and GTP similarly well, with nanomolar affinity. Mg2+ increases the affinity between p25rab3A and guanine nucleotides by 3- and 7-fold for GTP and GDP respectively, primarily by drastically decreasing the nucleotide off-rates. The Mg2+ binding affinity to p25rab3A. [alpha 32P]GDP was determined to be about 4 microns using entrapment of [alpha-32P]GDP as a measure of Mg2+ binding. At a Mg2+ concentration of 11 mM. GTPase activity was rate-limited by the GDP off-rate. Surprisingly, at a Mg2+ concentration of 80 nM. GTPase activity was comparable with that in the presence of excess Mg2+. In this case, kcat. was rate-limiting. At Mg2+ concentrations below 10 nM there was no detectable GTPase activity, indicating that Mg2+ is required for the GTPase activity of p25rab3A.

Animals↗

PubMed1992

The geranylgeranyl moiety but not the methyl moiety of the smg-25A/rab3A protein is essential for the interactions with membrane and its inhibitory GDP/GTP exchange protein.

The smg-25A/rab3A protein (smg p25A), a member of the small GTP-binding protein superfamily, has a C-terminal structure of Cys-Ala-Cys which is post-translationally processed: both cysteine residues are geranylgeranylated followed by the carboxyl methylation of the C-terminal cysteine residue. We reported previously that this posttranslational processing is essential for the interactions of smg p25A with membrane and its inhibitory GDP/GTP exchange protein, named smg p25A GDP dissociation inhibitor (GDI). In this study, we examined which posttranslational modification of smg p25A is necessary for these interactions. The smg p25A which was not posttranslationally processed was produced in Escherichia coli and purified. This protein was then geranylgeranylated at both of the 2 cysteine residues by use of a bovine brain geranylgeranyltransferase in a cell-free system (recombinant smg p25A-GG). By use of this recombinant smg p25A-GG, its membrane-binding activity and its sensitivity to smg p25A GDI were compared with those of the fully posttranslationally processed form of bovine brain smg p25A (smg p25A-GG-Me) and the posttranslationally unprocessed form of bacterial smg p25A (recombinant smg p25A). The membrane-binding activity and sensitivity to smg p25A GDI were similar between the recombinant smg p25A-GG and smg p25A-GG-Me, although recombinant smg p25A lacked both activities. These results indicate that the geranylgeranyl moiety of smg p25A is essential and sufficient for its interactions with membrane and smg p25A GDI and that the methyl moiety is not essential for these interactions.

Amino Acid Sequence↗

PubMed1992

Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A.

The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.

Animals↗

PubMed1992

Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells.

There is evidence that the rab class of low molecular weight GTP-binding proteins is involved in vesicular transfer from endoplasmic reticulum to Golgi and between Golgi cisternae. To determine whether similar proteins play a role in regulated exocytosis, the effects of synthetic peptides derived from low molecular weight GTP-binding proteins on catecholamine secretion from digitonin-permeabilized chromaffin cells were investigated. The synthetic peptides represent the putative effector-binding domains of the rab, ras and ral classes of low molecular weight GTP-binding proteins and correspond to ras(33-48). Two rab peptides but neither a ras nor a ral peptide enhanced Ca(2+)-dependent secretion by approximately 30%. Maximal secretion in response to Ca2+ was increased. The enhancement was not blocked by the pseudosubstrate inhibitor of protein kinase C, PKC(19-31), thus indicating that activation of protein kinase C was not responsible for the enhancement of secretion. Similarly a rab peptide but neither a ras nor a ral peptide enhanced GppNHp-induced secretion 30-70%. The peptides had little or no effect in the absence of Ca2+ or GppNHp. The data are consistent with a protein of the rab class playing a role in regulated exocytosis.

Adenosine Triphosphate↗

PubMed1992

Purification of squid synaptic vesicles and characterization of the vesicle-associated proteins synaptobrevin and Rab3A.

Two proteins associated with mammalian synaptic vesicles, the integral membrane protein synaptobrevin and the GTP-binding protein rab3A, are identified in squid nervous tissue using Western blotting and subcellular fractionation of synaptosomes. They both copurify with synaptic vesicles prepared from squid optic lobe. Synaptobrevin (18.1 kDa) is present at high levels in synaptic terminals but at very low levels in axon. Rab3A (27.5 kDa) is a member of the rab family of low-molecular weight GTP-binding proteins which regulates vesicle traffic in secretory and endocytic processes. As resolved with 2-dimensional gels, squid neurons contain at least 16 GTP-binding species (19-29 kDa), and most of these are present in both soluble and particulate fractions. The 24 kDa class of GTP-binding proteins is highly enriched in axonal transport organelles. The characterization of synaptobrevin and rab3A in squid synaptic vesicles extends their known distributions to invertebrates and points to a fundamental importance of these proteins in neurotransmitter release.

Animals↗

PubMed1992

Exocytotic fusion is activated by Rab3a peptides.

Studies of intracellular traffic in yeast and mammalian systems have implicated members of the Rab family of small GTP-binding proteins as regulators of membrane fusion. We have used the patch clamp technique to measure exocytotic fusion events directly and investigate the role of GTP-binding proteins in regulating exocytosis in mast cells. Intracellular perfusion of mast cells with GTP-gamma S is sufficient to trigger complete exocytotic degranulation in the absence of other intracellular messengers. Here we show that GTP is a potent inhibitor of GTP-gamma S-induced degranulation, indicating that sustained activation of a GTP-binding protein is sufficient for membrane fusion. We have found that synthetic oligopeptides, corresponding to part of the effector domain of Rab3a, stimulate complete exocytotic degranulation, similar to that induced by GTP-gamma S. The response is selective for Rab3a sequence and is strictly dependent on Mg2+ and ATP. This suggests that sustained activation of a Rab3 protein causes exocytotic fusion. The peptide response can be accelerated by GDP-beta S, suggesting that Rab3a peptides compete with endogenous Rab3 proteins for a binding site on a target effector protein, which causes fusion on activation.

Amino Acid Sequence↗

PubMed1992

Rab geranylgeranyl transferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys.

Rab proteins are membrane-bound prenylated GTP-binding proteins required for the targeted movement of membrane vesicles from one organelle to another. In the current paper we have characterized and purified an enzyme that attaches geranylgeranyl residues to Rab proteins that bear the COOH-terminal sequence Cys-X-Cys (such as Rab3A) and Cys-Cys (such as Rab1A). This enzyme is designated Rab geranylgeranyl transferase (Rab GG transferase). At high salt concentrations, Rab GG transferase from rat brain cytosol separates into two components, designated A and B, both of which are required for activity. We purified Component B to apparent homogeneity and found that it contains two peptides of 60 and 38 kDa. The purified Rab GG transferase did not attach geranylgeranyl to p21H-ras-CVLL, which is prenylated by a GG transferase of the CAAX type that resembles the CAAX farnesyltransferase. Rab GG transferase was strongly inhibited by Zn2+, a cation that is absolutely required by farnesyltransferase. The Rab GG transferase was also inhibited by NaCl concentrations in excess of 100 mM. Together with previous data, the current findings indicate that mammalian cells possess at least three protein prenyltransferases (CAAX farnesyltransferase, CAAX GG transferase, and Rab GG transferase) that are specific for different classes of low molecular weight GTP-binding proteins and other proteins.

Alkyl and Aryl Transferases↗

PubMed1992

Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A.

Two cellular factors have been described, Rab3A-GAP (GTPase-activating protein) and Rab3A-GRF (guanine nucleotide releasing factor) which, respectively, accelerate the intrinsic GTPase activity of, or the rate of dissociation of GDP from, the Ras-related GTP-binding protein, p25 Rab3A. Mutational analysis of p25 Rab3A was undertaken to define amino acid residues important for interaction with these factors. Mutations in residues 51-59, which correspond to the effector domain of p21 Ras, completely abolished sensitivity of p25 Rab3A to Rab3A-GRF and decreased the affinity of p25 Rab3A for Rab3A-GRF. Surprisingly, only one mutant in this region was Rab3A-GAP-insensitive, while the others retained partial, complete, or significantly increased GAP responsiveness. Mutations in the first G-domain had only modest effects on intrinsic GTPase activity and little effect on either Rab3A-GRF or Rab3A-GAP interactions. Truncation of 34 residues from the carboxyl terminus had no effect Rab3A-GAP sensitivity but facilitated Rab3A-GRF stimulation. Mutation T36N, analogous to the dominant inhibitory mutation T17N in Ras, which has been hypothesized to sequester an upstream activator of Ras, conferred a 10-fold higher affinity upon p25 Rab3A for Rab3A-GRF.

Animals↗

PubMed1992

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling.

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.

Adaptor Proteins, Vesicular Transport↗

PubMed1992

A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.

In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.

Amino Acid Sequence↗

PubMed1992

Expression of the ras-related rab3a gene in insulinoma-derived cell lines.

This study was designed to search for the expression of the small-molecular-weight GTP-binding protein rab3a in endocrine pancreatic cell lines. Total RNA was isolated from five different cell lines (RINm5F, RIN 104836, beta-TC1, HIT-15, and INRI-G9) and from whole rat brain. The expression of rab3a was analyzed by Northern blots. Similar as in brain two transcripts of 1300 and 1800 bp were detected in RIN-cells at low stringency conditions with the predominant signal at 1300 bp. At high stringency the stronger signal was at 1800 bp. When a 300 bp PstI fragment derived from the coding region of rab3a was utilized as probe the 1800 bp signal was predominant under each condition. Only a faint band at 1800 bp occurred in preparations from beta TC1-cells and no signal at all was found in HIT-15 and INRI-G9-cells. In conclusion, rab3a is expressed in rat insulin-releasing insulinoma-derived RIN-cells with a specific 1800 bp transcription product.

Animals↗

PubMed1992

Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction.

smg p25A is a small G protein which has been suggested to regulate neurotransmitter release from the synapses. We investigated here the ultrastructural localization of this small G protein in the rat neuromuscular junction by an immunoperoxidase method. The results showed that smg p25A was distributed non-uniformly on the presynaptic plasma membrane and among the synaptic vesicles with the focal accumulation on the discrete presynaptic sites which corresponded to the active zones, the regions of the presynaptic plasma membrane specialized for the exocytosis of the synaptic vesicles. This unique distribution of smg p25A suggests that it plays an important role in the attachment and fusion of the synaptic vesicles with the active zones.

Animals↗

PubMed1992

Distribution of smg p25A and smg p21s, ras p21-like guanine nucleotide-binding proteins, in the rat stomach.

Existence and distribution of small guanine nucleotide binding proteins (G proteins) such as smg p25A, smg p21s, and ras p21s were examined in the rat gastric mucosa. By immunoblot analysis using the specific monoclonal antibodies against smg p25A and ras p21 and the anti-smg p21 antiserum, smg p25A and smg p21s were detected in the gastric mucosa, while ras p21s were not detected. In immunocytochemical studies, moderate fluorescence of smg p25A was homogenously seen in the cytoplasmic portions of chief cells and surface epithelial cells. smg p21 immunoreactivity was detected in glandular cells and submucosal muscle layer in the mucosa. On the other hand, the cells constituting gastric glands were unstained with the anti-ras p21 monoclonal antibody, RASK-4. These results suggest that smg p25A and smg p21s exist as in other tissues and might exert their specific actions in the rat gastric mucosa.

Animals↗

PubMed1992

Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells.

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.

Adenosine Diphosphate Ribose↗

PubMed1992

An autosomal homologue of the choroideremia gene colocalizes with the Usher syndrome type II locus on the distal part of chromosome 1q.

Employing the mouse homologue of the human choroideremia cDNA as a probe, we have identified a homologous human gene. The consensus cDNA of this gene, designated human choroideremia-like (hCHML) gene, encompasses an open reading frame of 1968 base pairs. The deduced polypeptide of hCHML displays several regions of homology to smg p25A GDI, a bovine protein known to regulate the GDP/GTP exchange of the GTP-binding protein smg p25A. hCHML is located at 1q31-qter, a chromosomal region which, by means of linkage analysis, was previously shown to carry a gene locus for Usher syndrome type II. The colocalization of hCHML and Usher syndrome type II, as well as the clinical similarities between choroideremia and Usher syndrome type II, make hCHML a candidate gene for this disorder.

Amino Acid Sequence↗

PubMed1992

Localization of the ras-like rab3A protein in the adult rat brain.

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e., dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, regions of cerebral cortex and the cerebellum exhibited striking laminar distributions of rab3A immunoreactivity. In the hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentate granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral cortical hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.

Animals↗

PubMed1992

Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide.

In a previous study we demonstrated that a homeobox peptide corresponding to the 60 amino acid long DNA-binding region of the Drosophila antennapedia homeo-protein was capable of crossing the plasma membrane of cells in culture. This finding has led us to investigate whether chimeric molecules encompassing the homeobox would behave in a similar manner. We demonstrate here that a peptide of 93 amino acids composed of the homeobox and of the C terminus of Rab3, a small GTP-binding protein, crosses the membrane of myoblasts, myotubes and neurons and is conveyed to their nuclei. This transport is highly efficient, is observed in all the cells present in the culture and occurs at 37 degrees C and 12 degrees C without quantitative peptide degradation. Beyond its theoretical implications for our current views on cellular interactions, this finding could have technical repercussions on the development of drugs with intracellular targets.

Animals↗