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Results for “rab2 GTP-Binding Protein”

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At least 19 recordsLinked to original sources

PubMed1992

GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.

We have examined the role of ras-related rab proteins in transport from the ER to the Golgi complex in vivo using a vaccinia recombinant T7 RNA polymerase virus to express site-directed rab mutants. These mutations are within highly conserved domains involved in guanine nucleotide binding and hydrolysis found in ras and all members of the ras superfamily. Substitutions in the GTP-binding domains of rab1a and rab1b (equivalent to the ras 17N and 116I mutants) resulted in proteins which were potent trans dominant inhibitors of vesicular stomatitis virus glycoprotein (VSV-G protein) transport between the ER and cis Golgi complex. Immunofluorescence analysis indicated that expression of rab1b121I prevented delivery of VSV-G protein to the Golgi stack, which resulted in VSV-G protein accumulation in pre-Golgi punctate structures. Mutants in guanine nucleotide exchange or hydrolysis of the rab2 protein were also strong trans dominant transport inhibitors. Analogous mutations in rab3a, rab5, rab6, and H-ras did not inhibit processing of VSV-G to the complex, sialic acid containing form diagnostic of transport to the trans Golgi compartment. We suggest that at least three members of the rab family (rab1a, rab1b, and rab2) use GTP hydrolysis to regulate components of the transport machinery involved in vesicle traffic between early compartments of the secretory pathway.

Amino Acid Sequence↗

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↗

PubMed1991

Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

Animals↗

PubMed1992

Detection and characterization of mRNA and proteins encoded by human rab2 low molecular weight GTP-binding protein gene.

Three RNA species of 3.5, 2.4 and 1.4 kb were detected in all human hematopoietic, fibroblastic and tumor cell lines examined, as the mRNA of human rab2 gene presumably involved in intracellular transport. The mRNA differed in the length of the 3'-non-translated regions due to termination and/or processing at the alternative polyadenylation sites. The 1.4-kb rab2 RNA was predominant in Molt-4 and U-937 of mononuclear cell origin, while the 2.4-kb RNA was dominant in the other cells examined. The degradation of all rab2 RNA was similar and as slow as beta-actin RNA. The rab2 proteins were ubiquitously detected in the human cells as two phosphorylated peptides of major pp24rab2 and minor pp25rab2. pp25rab2 was slightly more phosphorylated than pp24rab2. Both rab2 proteins were abundantly detected in neural PC12 and NB3 cells, and mostly pp24rab2 was produced in the other human and rodent cells.

Animals↗

PubMed1992

p23rab2, a ras-like GTPase with a -GGGCC C-terminus, is isoprenylated but not detectably carboxymethylated in NIH3T3 cells.

With the development of a specific anti-rab2 antiserum, p23rab2, a ras-like GTPase with a -GGGCC C-terminus, has been localized mainly to the particulate (P100) fraction in NIH3T3 cells, although a small amount of this protein also appears in the soluble fraction. The endogenous p23rab2 is isoprenylated in intact cells, and recombinant murine p23rab2 is also isoprenylated in an in vitro system using reticulocyte lysate. Both the cytosolic and membrane-bound forms of p23rab2 are isoprenylated in intact cells. Recombinant p23rab2 is specifically geranylgeranylated at one or both of the C-terminal cysteine residues in the in vitro system. Blocking isoprenoid synthesis with lovastatin results in an accumulation of a totally cytosolic unisoprenylated form, indicating that isoprenylation is a prerequisite for membrane association of p23rab2. Surprisingly, unlike p21ras and p25rab3A, no carboxymethylation of p23rab2 is detectable in either the soluble or particulate fractions.

3T3 Cells↗

PubMed1992

Identification of rab2 as a tubulovesicle-membrane-associated protein in rabbit gastric parietal cells.

Rab proteins, which are ras-like low-molecular-mass GTP-binding proteins, are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. Previously, we reported a 23 kDa tubulovesicle-associated GTP-binding protein in rabbit gastric parietal cells [Basson, Goldenring, Tang, Lewis, Padfield, Jamieson & Modlin (1991) Biochem. J. 279, 43-48]. The major component of the 23 kDa protein is now identified as rab2. Rab2 was co-localized in tubulovesicle membranes from parietal cells. Consistent with GTP-binding activity (as documented before), upon maximal stimulation of parietal cells, rab2 immunoreactivity was redistributed from a 50,000 g to a 4000 g subcellular membrane fraction. The tubulovesicle-associated rab2 behaved as an integral membrane protein, since both 0.5 M-NaCl and 0.1 M-carbonate extraction failed to remove the protein from the tubulovesicle membrane. Utilizing a PCR the rab2 cDNA sequence from rabbit parietal cells was obtained, and it showed only one amino acid difference compared with the human sequence. The results of the present study provide strong evidence that parietal cells possess a rab2 protein which is tightly associated with tubulovesicle membranes.

Adenosine Triphosphatases↗

PubMed1992

Overexpression of the ras-related rab2 gene product in peripheral blood mononuclear cells from patients with hematological and solid neoplasms.

The rab2 gene product belongs to the rab branch of the ras-related GTP-binding protein family. The rab2 gene mRNA analysis in 70 tumor samples from various origins showed no obvious difference between malignant tissues and their normal counterparts. However, an over-expression has been observed at the RNA or protein levels in peripheral blood mononuclear cells from the nine patients with Sézary syndromes studied. Subsequent investigations in mycosis fungoides and solid tumor patients allowed us to link these high protein levels to immune reactive rather than to malignant cells. Circulating monocyte and lymphocyte populations from cancer patients are under analysis to correlate the Rab2 protein overexpression to a peculiar subset of cells.

Adult↗

PubMed1992

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.

Animals↗

PubMed1989

The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion.

Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library. They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily. The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized. Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities. H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart. The other proteins share approximately 40% homology with YPT1p and SEC4p. The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding. Human rab proteins were produced in Escherichia coli. Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents. All six proteins bind GTP and exhibit GTPase activities. A possible involvement of the rab proteins in secretion is discussed.

Adrenal Gland Neoplasms↗

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↗

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↗

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↗

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↗

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↗