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Biomedical subjects

B Goud

Publications and source records attributed to B Goud.

At least 73 records · Page 4Linked to original sources
PubMed1992

The low molecular weight guanosine triphosphate-binding protein Rab6p associates with distinct post-Golgi vesicles in Torpedo marmorata electrocytes.

The Rab genes have recently been cloned and sequenced in mammals, and their products represent good candidates for low molecular weight guanosine triphosphate-binding proteins involved in the regulation of intracellular transport of vesicles in higher eukaryotes. Remarkably, each of the Rab proteins appears to be associated with a distinct step of either the exocytic or endocytic pathway. In particular, Rab6p has been localized to the outermost Golgi cisternae in normal rat kidney cells, where its function remains unclear. In this work, we have carried out a series of immunocytochemical analyses of the subcellular distribution of Rab6p in a polarized cell, the electrocyte of Torpedo marmorata, to elucidate the molecular mechanisms involved in the sorting and targeting of synaptic proteins. We report that, aside from its Golgi localization, the bulk of Rab6p associates with clusters of post-Golgi vesicles, primarily those located at the cytoplasmic face of the innervated membrane of the electrocyte. Consequently, Rab6p presents a polarized distribution in this cell. Furthermore, we show that this distribution is dependent upon the integrity of the microtubule network of the electrocyte. These data are coherent with the notion that Rab6p is involved in the regulation of membrane traffic from the trans-Golgi network to the innervated plasma membrane, delivering, by way of a microtubule-based organelle transport mechanism, synaptic proteins to their appropriate locations.

Animals↗
PubMed1992

Small GTP-binding proteins as compartmental markers.

Each intracellular compartment involved in the biosynthetic/secretory pathway of eukaryotic cells bears at its surface at least one small GTP-binding protein. Most of them belong to a distinct branch of the p21ras superfamily, the Sec4/Ypt1/rab family. Other proteins are members of the ARF family. They play a key role in the regulation of budding and targeting/fusion events occurring during protein transport.

Amino Acid Sequence↗
PubMed1992

The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach.

A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exocytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20 degrees C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.

Biological Transport↗
PubMed1991

The small GTP-binding protein rab4 is associated with early endosomes.

Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. We demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of approximately 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing 125I-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. We also localized rab4p to transferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4 cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular transport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

Animals↗
PubMed1991

Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2.

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.

Amino Acid Sequence↗
PubMed1991

Association of the Ras-antagonistic Rap1/Krev-1 proteins with the Golgi complex.

Ras oncogenes encode 21-kDa GTP-binding proteins that are capable of transforming immortalized cells in culture. Ras proteins are bound to the inner face of the plasma membrane by their C-terminal extremity and are thought to transmit their mitogenic signals via an "effector" domain spanning amino acids 32-42. Two ras-related human genes rap1A and rap1B encode 95% homologous 21-kDa proteins that share with Ras p21 the same effector domain and a similar C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid; also known as a CAAX sequence). The product of the rap1A gene is identical to that of the Krev-1 cDNA, whose overexpression is capable of reverting the phenotype of Ki-ras-transformed NIH 3T3 cells. Antibodies that do not cross-react with Ras and other Ras-related proteins were obtained by immunizing rabbits with a peptide encompassing residues 121-137 of Rap1 proteins. These antibodies were used to investigate the subcellular localization of Rap1 proteins by indirect immunofluorescence and fractionation techniques. Rap1 proteins were found to be tightly bound to cellular membranes. They did not colocalize with Ras proteins on the plasma membrane and were discovered to be associated with the Golgi complex.

Amino Acid Sequence↗
PubMed1991

Subcellular distribution of clathrin in cultured hypothalamic neurons.

The subcellular distribution of clathrin has been examined in developing hypothalamic neurons cultured in a chemically defined medium up to synapse formation (12-13 days in vitro) and exposed, or not, to a depolarizing concentration of KCl (60 mM for 3 min) followed, or not, by a return to control KCl concentration (3 mM KCl for 3 min). Previous studies have shown that such treatments induce in synaptic boutons a rapid vesicle depletion followed by massive restoration. Using an enzyme immunoassay, we have compared the relative proportion of assembled and unassembled pools of clathrin as a function of exposure to depolarizing or repolarizing concentrations of KCl. In parallel we have localized clathrin at the electron microscopic level using immunoperoxidase. Clathrin concentration in culture is lower (0.36 vs 0.75%) and the proportion of unassembled clathrin is much higher than in the adult brain (82 vs 14%). These proportions were not affected by depolarizing or repolarizing treatments. Morphologically clathrin was exclusively detected in two neuron compartments: perikarya and synaptic boutons. In perikarya clathrin was localized as a thick coat on plasma membrane coated pits and in the Golgi zone on coated buds and vesicles, presumably located in a trans compartment. In synaptic boutons clathrin immunoreaction was found as an irregular thin rim around synaptic vesicles, whatever the polarization state of the cells, but coated vesicles were extremely rare. Taken together these findings raise the problem of the functional meaning and localization of the large unassembled pool of clathrin in such neurons and question its role in vesicular traffic in synaptic boutons.

Animals↗
PubMed1991

The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.

Blotting, Western↗
PubMed1991

Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation.

Rab is a newly identified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Ypt1p involved in the regulation of the secretory pathway. Mature phagocytes display functions requiring organized intracellular traffic and, for this reason, we questioned whether phagocyte differentiation could correlate with the increased expression of rab proteins. Rabbit antisera raised against the recombinant proteins rab1Ap, 2p, 4p, and 6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937. When these cells were induced to differentiate into monocyte/macrophage-like cells displaying functional characteristics of a normal phagocyte, rab1Ap, 2p, 4p, and 6p were increased and this correlated with an increase in the rab transcripts. Using a rab5 probe, we also observed an increased expression of the rab5 gene in differentiated cells. Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased expression of the rab proteins. Rab proteins were also detected in human neutrophils and in guinea pig alveolar macrophages. As degranulation is one of the phagocyte functions acquired in the late stage of differentiation, we investigated whether rab proteins would be involved in this process. Although rab proteins were tightly membrane bound, none of them was detected in the specific or azurophil granules purified from human neutrophils. The increased expression of rab proteins in mature phagocytes suggests that they may promote functions highly developed in these cells.

Animals↗
PubMed1990

Small GTP-binding protein associated with Golgi cisternae.

Eukaryotic cells seem to use GTP hydrolysis to regulate vesicular traffic in exocytosis and endocytosis. The best evidence for this comes from studies on the yeast Saccharomyces cerevisiae that have identified two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, which control distinct stages of the secretory pathway. In mammalian cells the effects of a non-hydrolysable GTP analogue, GTP-gamma S, on different transport events have suggested that they also have proteins functionally related to yeast Sec4p and Ypt1p. The rab genes have recently been cloned and sequenced for rat and human and their proteins have highly conserved domains in common with Sec4p and Ypt1p (including a putative effector binding site). They are therefore good candidates for GTP-binding proteins involved in intracellular transport in mammalian cells. One of the Rab proteins (Rab1p) is the mammalian counterpart of Ypt1p (ref. 13). Here we report the localization of the protein Rab6p to the Golgi apparatus in several cell types. By immunolabelling and electron microscopy, Rab6p appears to be concentrated predominantly on the medial and trans cisternae and distributed over their entire surface.

Blotting, Western↗
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↗
PubMed1990

Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity.

Since human immunodeficiency virus (HIV) nef has been suggested to exert regulatory effects on HIV long terminal repeat (LTR) activity, we transiently transfected HIV LTR chloramphenicol acetyltransferase or luciferase expression vectors into a human astrocytoma clone (U-373nef) that constitutively expresses the HIV nef gene. In these cells, basal HIV LTR activity, as well as tumor necrosis factor-induced or tat-driven activity, was similar to that in control cells. Lack of any detectable effect of HIV nef on LTR activity was not the result of mutations in integrated nef DNA, as was shown by polymerase chain reaction. These data suggest that the role of nef in HIV genome transcription does not necessarily involve a direct influence on HIV LTR activity.

Astrocytoma↗
PubMed1990

Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus.

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.

Animals↗
PubMed1989

Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic.

Mutant alleles of SEC4, an essential gene required for the final stage of secretion in yeast, have been generated by in vitro mutagenesis. Deletion of the two cysteine residues at the C terminus of the protein results in a soluble non-functional protein, indicating that those two residues are required for normal localization of Sec4p to secretory vesicles and the plasma membrane. A mutant allele of SEC4 generated to mimic an activated, transforming allele of H-ras, as predicted, does not bind GTP. The presence of this allele in cells containing wild-type SEC4 causes a secretory defect and the accumulation of secretory vesicles. The results of genetic studies indicate that this allele behaves as a dominant loss of function mutant and as such prevents wild-type protein from functioning properly. We propose a model in which Sec4p cycles between an active and an inactive state in order to mediate the fusion of vesicles to the plasma membrane.

Alleles↗
PubMed1989

Fractionation of yeast organelles.

In summary, organelles of the secretory pathway can be effectively separated from one another using differential centrifugation followed by sucrose density gradient fractionation of wild-type or vesicle-accumulating mutant yeast cells. Up to 10-fold enrichment of the plasma membrane fraction is obtained, and resolution of the peak fractions of several organelles allows one to localize specific proteins to particular components of the pathway. Additionally, a highly purified population of constitutive secretory vesicles can be isolated from the 100,000 g membrane fraction of sec 6-4 cells on a Sephacryl S-1000 column. The success of this procedure is due to the homogeneous size of the vesicles and the high concentration of vesicles accumulated in the sec 6-4 cells. From other laboratories, methods have been described for the isolation of other organelles including the vacuole (Wiemken, 1975), plasma membrane (Tschopp and Schekman, 1983), and nuclei (Mann and Mecke, 1980), as well as an alternative procedure for the purification of secretory vesicles from yeast (Holcomb et al., 1987). For the localization of proteins to particular organelles the ability to lyse cells osmotically is an important improvement over the glass bead lysis procedure. The shear forces generated during glass bead lysis could potentially remove proteins from the surface of organelles that otherwise would be membrane-attached, causing them to appear soluble. Similarly, because the conditions required for stabilizing the association of a protein with a membrane can be quite variable depending on the lysis buffer, confirmation of localization using alternative schemes is prudent. With the advent of such techniques as confocal immunofluorescent microscopy and immunoelectron microscopy, effective methods for confirming localizations are becoming available.

Cell Fractionation↗
PubMed1988

A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast.

SEC4, one of the 10 genes involved in the final stage of the yeast secretory pathway, encodes a ras-like, GTP-binding protein. In wild-type cells, Sec4 protein is located on the cytoplasmic face of both the plasma membrane and the secretory vesicles in transit to the cell surface. In all post-Golgi blocked sec mutants, Sec4p is predominantly associated with the secretory vesicles that accumulate as a result of the secretory block. Sec4p is synthesized as a soluble protein that rapidly (t1/2 less than or equal to 1 min) and tightly associates with secretory vesicles and the plasma membrane by virtue of a conformational change of a covalent modification. These data suggest that Sec4p may function as a "G" protein on the vesicle surface to transduce an intracellular signal needed to regulate transport between the Golgi apparatus and the plasma membrane.

Cell Membrane↗
PubMed1988

Antibody-mediated binding of a murine ecotropic Moloney retroviral vector to human cells allows internalization but not the establishment of the proviral state.

We have attempted to introduce a neomycin-resistance gene, recombined in a murine (ecotropic) Moloney retrovirus, into HEp2 human cells which are resistant to infection by this retrovirus. To allow the binding of the retroviral vector on HEp2 cells, we have tested several antibody constructions between two monoclonal antibodies, one (mAb 5E9) directed against the human transferrin receptor and the other (mAb 615) directed against the gp70 envelope viral protein. The crosslinking of mAbs 5E9 and 615 by a sheep anti-murine kappa light chain antibody allowed the binding of virus on HEp2 cells. After incubation at 37 degrees, virus was internalized by HEp2 cells. However, no neomycin-resistant colonies of HEp2 cells have been obtained under various conditions. Our results suggest that binding and internalization of a retrovirus are not sufficient for establishment of the proviral state in cells which do not express a specific receptor.

Antibodies, Monoclonal↗