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M Zerial

Publications and source records attributed to M Zerial.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rab11 regulates recycling through the pericentriolar recycling endosome.

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

Animals↗
PubMed1996

Insulin induces a change in Rab5 subcellular localization in adipocytes independently of phosphatidylinositol 3-kinase activation.

We investigated whether Rab5, a small guanosine triphosphatase that regulates early endocytic transport in different cell types is involved in the insulin-regulated endocytic pathways in adipocytes. Rab5 was detected in freshly isolated adipocytes and 3T3-L1 adipocytes, but its expression level was not markedly increased with adipocyte differentiation. After subcellular fractionation of adipocytes incubated in the absence of insulin, Rab5 was found to be abundant in plasma membrane and cytosol, but was also present in high and low density microsomes. This subcellular distribution was compatible with a role in early endocytosis. When cells were incubated with insulin, the concentration of Rab5 decreased by about 50% in the internal compartments. In contrast to Rab4, which also leaves the low density microsomes in response to insulin, Rab5 was not found in Glut4-containing vesicles purified by immunoadsorption on antibodies to Glut4. When adipocytes were treated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, the effect of insulin on Rab5 movement was not affected, whereas the insulin-induced movements of Rab4 and Glut4 were abolished. In parallel, wortmannin inhibited the increase in horseradish peroxidase uptake induced by insulin, an index of fluid phase endocytosis, but did not prevent the endocytosis of the glucose transporters. As a whole, our results suggest that Rab5 is not involved in insulin-stimulated Glut4 exocytosis. These results are compatible with the postulated role of Rab5 in the endocytotic pathway, at a step that does not require phosphatidyl-inositol 3-kinase activation.

3T3 Cells↗
PubMed1995

The Rab protein family: genetic mapping of six Rab genes in the mouse.

Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking. Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice. To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an inter-subspecific backcross [C57BL/6J-bgJ x (C57BL/6J-bgJ x CAST/Ei)F1] segregating for the bg locus. Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F1 and C57BL/6J chromosomal DNA with the coding sequences of Rab genes. These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice. Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively. Although the results exclude these loci as candidates for bg, they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively.

Animals↗
PubMed1995

Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion.

We have identified a novel 100 kDa coiled-coil protein, rabaptin-5, that specifically interacts with the GTP form of the small GTPase Rab5, a potent regulator of endocytic transport. It is mainly cytosolic, but a fraction colocalizes with Rab5 to early endosomes. Expression of a GTPase-deficient Rab5 mutant enhances the binding of rabaptin-5 to enlarged endosomes. Overexpression of rabaptin-5 alone is sufficient to promote expansion of early endosomes. Rab5 recruits rabaptin-5 to purified early endosomes in a GTP-dependent manner, demonstrating functional similarities with other members of the Ras superfamily. Immunodepletion of rabaptin-5 from cytosol strongly inhibits Rab5-dependent early endosome fusion. Rabaptin-5 is thus a Rab effector required for membrane docking and fusion.

Amino Acid Sequence↗
PubMed1995

Co-operative regulation of endocytosis by three Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

Amino Acid Sequence↗
PubMed1995

A GDP/GTP exchange-stimulatory activity for the Rab5-RabGDI complex on clathrin-coated vesicles from bovine brain.

Small GTPases of the Rab family are key regulators of intracellular transport. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecting these compartments. Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibitor (RabGDI). Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form. In this paper we have investigated whether Rab5, which regulates the clathrin-coated vesicle-mediated pathway of endocytosis, can directly associate with the membrane of clathrin-coated vesicles (CCV) purified from bovine brain in vitro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV. Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociation of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP. The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone. However, maximal GDP dissociation stimulation required the presence of RabGDI, suggesting that the factor(s) responsible for the membrane association and GDP/GTP exchange of Rab5 recognize the protein complexed to RabGDI. These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.

Animals↗
PubMed1995

Isolation of a murine cDNA clone encoding Rab19, a novel tissue-specific small GTPase.

Using a rapid amplification of cDNA ends (RACE) cloning approach, we have isolated a cDNA clone encoding Rab19, a novel small GTPase of the Rab subfamily contained within partial sequences previously described [Chavrier et al., Gene 112 (1992) 261-264]. Northern blot analysis of the distribution of the rab19 mRNA in various adult mouse tissues and NIH 3T3 fibroblasts revealed that rab19 is expressed in a tissue-specific manner. The rab19 transcript was detected at high levels in intestine, lung and spleen, and at a lower level in kidney. In contrast, liver, brain, heart and NIH 3T3 fibroblasts contain only very little or no detectable rab19 mRNA. Therefore, Rab19 is likely to represent a novel tissue- or cell type-specific small GTPase.

Amino Acid Sequence↗
PubMed1995

Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway.

Ypt51p, a small GTPase of Saccharomyces cerevisiae, has been previously identified as a structural homolog of mammalian Rab5. Although disruption analysis revealed that the protein is required for endocytic transport and for vacuolar protein sorting, the precise step controlled by Ypt51p was not determined. In this work we show that by heterologous expression in animal cells Ypt51p was targeted to Rab5-positive early endosomes and stimulated endocytosis. Furthermore, two Ypt51p mutants induced similar morphological alterations as the corresponding Rab5 mutants. Also in yeast cells Ypt51p was found to be required at an early step in endocytic membrane traffic, since alpha-factor accumulated in an early endocytic intermediate in the absence of Ypt51p. Cell fractionation analysis revealed cofractionation of Ypt51p with endocytic intermediates, while no association with the late Golgi compartment could be detected. Indirect immunofluorescence microscopy allowed us to morphologically identify the Ypt51p-containing compartment. Similar to the mammalian system larger Ypt51p-positive structures were revealed upon expression of Ypt51p Q66L. These structures were also positive for alpha-factor receptor and for carboxypeptidase Y, thus providing direct evidence for their endocytic nature and for the convergence of the vacuolar biosynthetic and endocytic pathways.

Animals↗
PubMed1994

Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes.

Rab proteins comprise a family of small GTPases that serve a regulatory role in vesicular membrane traffic. Geranylgeranylation of these proteins on C-terminal cysteine motifs is crucial for their membrane association and function. This post-translational modification is catalysed by rab geranylgeranyl transferase (Rab-GGTase), a multisubunit enzyme consisting of a catalytic heterodimer and an accessory component, named rab escort protein (REP)-1. Previous in vitro studies have suggested that REP-1 presents newly synthesized rab proteins to the catalytic component of the enzyme, and forms a stable complex with the prenylated proteins following the transfer reaction. According to this model, a cellular factor would be required to dissociate the rab protein from REP-1 and to allow it to recycle in the prenylation reaction. RabGDP dissociation inhibitor (RabGDI) was considered an ideal candidate for this role, given its established function in mediating membrane association of prenylated rab proteins. Here we demonstrate that dissociation from REP-1 and binding of rab proteins to the membrane do not require RabGDI or other cytosolic factors. The mechanism of REP-1-mediated membrane association of rab5 appears to be very similar to that mediated by RabGDI. Furthermore, REP-1 and RabGDI share several other functional properties, the ability to inhibit the release of GDP and to remove rab proteins from membranes; however, RabGDI cannot assist in the prenylation reaction. These data suggest that REP-1 is per se sufficient to chaperone newly prenylated rab proteins to their target membranes.

Alkyl and Aryl Transferases↗
PubMed1994

Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments.

Pathogenic strains of Helicobacter pylori cause progressive vacuolation and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, transferrin, and transferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by affinity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findings indicate that the vacuoles specifically originate from late endosomal compartments.

Adenine↗
PubMed1994

Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells.

In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes.

Amino Acid Sequence↗