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Results for “rab2 GTP-Binding Protein”

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PubMed1994

Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.

Animals↗

PubMed1993

Gene cloning and characterization of a GTP-binding Rab protein from mouse pituitary AtT-20 cells.

The Rab proteins constitute a family of Ras-like, low-molecular-weight GTP-binding proteins that are involved in vesicular transport in mammalian cells. Several members of this family have been localized to specific cellular compartments, indicating that they may control vesicular transfer between discrete portions of the endomembrane systems. To examine the complexity of the Rab proteins produced in cells with both regulated and constitutive secretory pathways, we used a PCR-based strategy to clone members of this gene family from rodent pituitary. Here, we report the identification and studies of one of the clones, RAB18. The full-length cDNA clone from a mouse pituitary AtT-20 cDNA library encodes a 23.5-kDa protein with four consensus GTP-binding domains. The deduced amino acid sequence is 42% and 40% identical to Rab2 and Sec4p, respectively, and contains sequence motifs characteristic of the Rab family. Northern blot analysis shows that the level of expression is high in the brain, moderate in the pituitary, and low in the liver. Rab18 may be involved in membrane traffic events during biogenesis and/or recycling steps of secretory vesicle components.

Amino Acid Sequence↗

PubMedDate not supplied

Small GTP-binding proteins in parietal cells: candidate modulators of parietal cell membrane dynamics.

The stimulated fusion of intracellular H/K-ATPase-containing tubulovesicles with a target canalicular membrane surface is central to the process of acid secretion. A super-family of small GTP-binding proteins (smGTPBPs) has been implicated in many aspects of intracellular dynamics and vesicle membrane trafficking. We have investigated the presence of smGTPBPs in isolated rabbit parietal cells. Parietal cells possess a number of smGTPBP species with molecular masses of 18-28 kDa. One 23 kDa smGTPBP has been localized to tubulovesicles and identified immunochemically as rab2. Rab2 redistributes during stimulation in concert with the movement of the H/K-ATPase. The results demonstrate that specific smGTPBPs are associated with the parietal cell secretory apparatus. Small GTP-binding proteins are important candidate regulators of parietal secretory membrane dynamics.

Animals↗

PubMed1996

Isolation of an additional soybean cDNA encoding Ypt/Rab-related small GTP-binding protein and its functional comparison to Sypt using a yeast ypt1-1 mutant.

We have previously reported the isolation of a gene from a soybean cDNA library encoding a Ypt/Rab-related small GTP-binding protein, Sypt. Here, we report the isolation of a second Ypt/Rab-related gene, designated Srab2, from the same soybean cDNA library. And we compare the in vivo function of the two soybean genes utilizing a yeast ypt1-1 mutant. The Srab2 gene encodes 211 amino acid residues with a molecular mass of 23 169 Da. The deduced amino acid sequence of the Srab2 is closely related to the rat (76%) and human (75%) Rab2 proteins, but it shares relatively little homology to Sypt (46%) and Saccharomyces cerevisiae ypt proteins (41%). Genomic Southern blot analysis using the cDNA insert of Srab2 revealed that it belongs to a multigene family in the soybean genome. The protein encoded by Srab2 gene, when expressed in Escherichia coli, disclosed a GTP-binding activity. The expression pattern of the Srab2 gene is quite different from that of the Sypt gene. The Srab2 gene is predominantly expressed in the plumule region, while expression was very low in the other areas in soybean seedlings. On the other hand, the Sypt mRNA is not detectable in any tissues of soybean seedlings grown in the dark. However, light significantly suppressed the Srab2 gene expression, but enhanced the transcript levels of the Sypt gene in leaf and, at even higher levels, in root tissues. When the Srab2 and Sypt genes are introduced separately into a S cerevisiae defective in vesicular transport function, the Srab2 gene cannot complement the temperature-sensitive yeast ypt1-1 mutation at all, in contrast to the Sypt gene. In conclusion, the difference of functional complementation of the yeast mutation together with differential expression of the two genes suggest that the in vivo roles of the Srab2 and Sypt genes may be different in soybean cells.

Amino Acid Sequence↗

PubMed1991

rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif.

Low molecular mass GTP-binding proteins encoded by the mammalian rab genes are found in membranes of the Golgi complex and endosomes, suggesting that they play a role in the movement of exocytic and endocytic vesicles. The basis for the membrane association of these proteins has not been defined. Herein, we demonstrate that terminal cysteine residues in the rab1B, rab2, and rab5 proteins undergo thioether modification by isoprenyl groups when these proteins are translated in vitro in the presence of a radiolabeled isoprenoid precursor, [3H]mevalonate. Results of gel permeation chromatography of the radiolabeled hydrocarbons suggest that these proteins are modified specifically by isoprenyl groups of the 20-carbon diterpene class, rather than the 15-carbon farnesyl class known to be involved in modification of ras proteins. The rab1 and rab2 proteins lack the carboxyl-terminal amino acid motif common to all previously identified isoprenylated proteins, i.e. CXXX, where X is an unspecified amino acid. Analysis of altered translation products generated by site-directed mutagenesis indicates that modification of rab1B protein requires an intact carboxyl-terminal sequence consisting of GGCC. This represents a new amino acid motif for isoprenylation.

Amino Acid Sequence↗

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↗

PubMed1991

rab5 controls early endosome fusion in vitro.

The small GTP-binding protein rab5 was previously localized on early endosomes and on the cytoplasmic face of the plasma membrane. Using a cell-free assay, we have now tested whether rab5 is involved in controlling an early endocytic fusion event. Fusion could be inhibited by cytosol containing the overexpressed mutant rab5lle133, which does not bind GTP on blots, and by antibodies against rab5, but not against rab2 or rab7. In contrast, fusion was stimulated with cytosol containing overexpressed wild-type rab5. Cytosols containing high levels of rab2 or mutant rab5 with the 9 carboxy-terminal amino acids deleted, which bind GTP on blots, had no effects. Finally, the inhibition mediated by anti-rab5 antibodies could be overcome by complementing the assay with the cytosol containing wild-type rab5, but not with the same cytosol depleted of rab5, nor with cytosol containing the rab5 mutants or rab2. These in vitro findings strongly suggest that rab5 is involved in the process of early endosome fusion.

Animals↗

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↗

PubMed1991

Hypervariable C-terminal domain of rab proteins acts as a targeting signal.

Mammalian cells express many ras-like low molecular mass GTP-binding proteins (rab proteins) that are highly homologous to the Ypt1 and Sec4 proteins involved in controlling secretion in yeast. Owing to their structural similarity and to their variety, rab proteins have been postulated to act as specific regulators of membrane traffic in exocytosis and endocytosis, and rab5 has been shown to be involved in early endosome fusion in vitro. In agreement with their postulated functions, all rab proteins studied so far have been found in distinct subcompartments along the exocytic or endocytic pathways. To define the region mediating their specific localization, we transiently expressed rab2, rab5 and rab7 hybrid proteins in BHK cells, and determined their intracellular localization by immunofluorescence confocal microscopy and subcellular fractionation. Here we present evidence that the highly variable C-terminal domain contains structural elements necessary for the association of rab proteins with their specific target membranes in the endocytic pathway.

Animals↗

PubMed1994

Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.

Amino Acid Sequence↗

PubMed1990

The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro.

The rab genes code for small GTP binding proteins that share with p21ras the ability to bind and hydrolyze GTP. They present significant sequence homologies with the products of YPT1 and SEC4, two small GTP binding proteins involved in the regulation of secretion in the yeast. Several rab genes are expressed in the developing and adult mouse brain. To test directly the possible involvement of these genes in neuronal differentiation, purified rab proteins produced in E. coli were introduced into neurons dissociated from E15 rat midbrain. The most striking effects were obtained with rab2 protein (rab2p). Compared with untreated cells, neurons loaded with rab2p presented an enhanced adhesion to the culture substratum. This phenomenon was visible 3 hr after seeding and was followed within 24 hr by a dramatic increase in neurite growth. Loading the same population of neurons with the products of four other rab genes either decreased neuronal adhesion and neurite growth or had no effect. These experiments suggest that the expression of rab2p plays an important role in neuronal differentiation.

Animals↗

PubMed1994

Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins.

The kinetic properties of MSS4 are studied in comparison with those of Rab3A GRF. MSS4 stimulates the dissociation of [3H]GDP from the lipid-modified and lipid-unmodified forms of Rab3A to the same extent, although Rab3A GRF is more effective on the lipid-modified form than on the lipid-unmodified form. Both MSS4 and Rab3A GRF are inactive on other Rab/Sec/Ypt family members including at least Rab2, Rab5, and Rab11. Rab GDI inhibits the MSS4 and Rab3A GRF effects on the lipid-modified form of Rab3A, but the doses of Rab GDI necessary for this inhibitory effect on Rab3A GRF are lower than those on MSS4. Moreover, Rab GDI slightly inhibits the Rab3A GRF effect on the lipid-unmodified form of Rab3A, but does not affect the MSS4 effect on the lipid-unmodified form of Rab3A. These results suggest that MSS4 and Rab3A GRF are different GDP/GTP exchange proteins for Rab3A.

GTP-Binding Proteins↗

PubMed1989

Developmental and regional expression of three new members of the ras-gene family in the mouse brain.

We have examined the expression in the mouse nervous system of three new members of the ras protooncogene family: rab1, rab2, and rab3. Each of these genes was transcribed into messenger RNAs with different molecular weights. These transcripts has specific developmental and regional patterns of expression. In particular, for the three genes, the ratio between the heavy and light mRNAs depended strongly on developmental stage and brain region. The use of pure neuronal and glial cultures revealed that the high molecular weight transcripts were enriched in neurons and that, in the case of rab2 and rab3, their expression increased with neuronal differentiation. These results are discussed considering the sequence identities between these genes and the yeast YPTI and sec-4 genes, which are known to be implicated in post-Golgi vesicular transport and cytoskeletal stabilization. We propose that the rab genes might be of importance in the regulation of these two processes within the developing and adult nervous system.

Aging↗

PubMed1995

Two-hybrid system screen with the small GTP-binding protein Rab6. Identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6.

Rab6 is a small GTP-binding protein that belongs to the Ras superfamily and is involved in intra-Golgi transport. Using a two-hybrid system screen of a mouse brain cDNA library, we have isolated several clones encoding proteins that interact with Rab6. Approximately 60% of the clones identified encoded a new mouse Rab GDP dissociation inhibitor (GDI) isoform. This GDI isoform is distinct from mouse mGDI-1 and mGDI-2, which have been characterized previously, and most likely represents the mouse counterpart of the rat Rab GDI beta isoform. In the two-hybrid system, GDI beta interacts with wild-type Rab6 and Rab5, but not with a GTP-bound Rab6 mutant, or a Rab6 mutant that cannot be post-translationally processed. We further examined whether mouse GDI beta is functional; we show that recombinant mouse GDI beta is able to remove several Rab proteins, including Rab1, Rab2, Rab4, and Rab6, from membranes. The identification of a third GDI isoform in mouse raised the question whether GDI genes belong to a larger multigenic family. We have shown, by Southern blot analysis of genomic DNA, that at least five GDI gene copies exist in both the mouse and rat genomes. In our two-hybrid screen, we have also characterized another clone that specifically interacts with Rab6. This clone was partially sequenced but shows no homology to known sequences. Finally, a third clone, interacting with both Rab5 and Rab6, also appears to encode a novel protein.

Amino Acid Sequence↗

PubMed1996

Rho proteins are localized with different membrane compartments involved in vesicular trafficking in anterior pituitary cells.

In order to explore the role of certain GTP binding proteins in the rat anterior pituitary, we have analyzed the subcellular distribution of the proteins rho and rab. They were found in both membrane and cytosolic fractions. Rab1 and rab2 were localized in both Golgi and endoplasmic reticulum (ER) membranes, while rab4 and rab6 were found in fractions enriched with Golgi and plasma membranes, implicating these proteins in the control of vesicular intracellular trafficking as described in other systems. Rab3 was localized like a fraction of synaptophysin, suggesting a role for rab3 in the targeting of "synaptic-like' microvesicles. We have identified three substrates of C. botulinum exoenzyme C3. A 26-kDa substrate with an isoelectric point (pI) of 5.2, probably rhoB, was localized in the lightest fractions such as rab3 and synaptophysin proteins. Two other 23-24 kDa substrates with pI of 5.5-5.8, probably rhoA and/or rhoC, were found in both fractions enriched with ER and secretory granules. Rho proteins have been implicated in the control of actin polymerization. Their localization in anterior pituitary suggests that rhoB could control the association of synaptic-like microvesicles and plasma membrane, and that rhoA/rhoC could play a role in secretory granule exocytosis; these two pathways being involved in cytoskeleton protein reorganisation in response to extracellular signals.

ADP Ribose Transferases↗

PubMed1994

Specific interactions of Mss4 with members of the Rab GTPase subfamily.

Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4-GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co-precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.

Animals↗

PubMed1990

Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments.

A set of 11 clones encoding putative GTP binding proteins highly homologous to the yeast YPT1/SEC4 gene products have been isolated from an MDCK cell cDNA library. We localized three of the corresponding proteins in mammalian cells by using affinity-purified antibodies in immunofluorescence and immunoelectron microscopy studies. One, the MDCK homolog of rab2, is associated with a structure having the characteristics of an intermediate compartment between the endoplasmic reticulum and the Golgi apparatus. The second, rab5, is located at the cytoplasmic surface of the plasma membrane and on early endosomes, while the third, rab7, is found on late endosomes. These findings provide evidence that members of the YPT1/SEC4 subfamily of GTP binding proteins are localized to specific exocytic and endocytic subcompartments in mammalian cells.

Amino Acid Sequence↗

PubMed1995

Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.

Adrenal Medulla↗