PubMed Health⌕ Search

Biomedical subjects

M Zerial

Publications and source records attributed to M Zerial.

At least 55 records · Page 3Linked to original sources
PubMed1994

Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.

Animals↗
PubMed1994

Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange.

The Rab GTPases function as specific regulators of membrane transport. The GTP/GDP cycle is believed to control shuttling of Rab proteins between the cytosol and organelle membranes. In vitro, Rab proteins are removed from membranes by a protein that inhibits GDP dissociation (rabGDI), which leads to formation of a cytosolic complex of Rab with the inhibitor protein. Here we use a purified Rab5-rabGDI complex in a permeabilized cell system to investigate how the cytosolic complexed form of Rab reassociates with the membrane. We find that exogenous Rab5 is correctly targeted and induces the formation of enlarged early endosomes, demonstrating that it is functionally active. Binding of Rab5 to the acceptor membrane is accompanied by release of the rabGDI protein into the cytosol. A transient GDP-Rab5 intermediate was detected which was subsequently converted into the GTP-bound form. Our results indicate that there is a multistep mechanism for the insertion of Rab5 into the membrane which is mediated by a guanine-nucleotide-exchange factor.

Animals↗
PubMed1994

Distinct structural elements of rab5 define its functional specificity.

Members of the rab family of small GTPases are localized to distinct cellular compartments and function as specific regulators of vesicle transport between organelles. Overexpression of rab5, which is associated with early endosomes and the plasma membrane, increases the rate of endocytosis [Bucci et al. (1992) Cell, 70, 715-728]. From sequence alignments and molecular modelling we identified structural elements that might contribute to the definition of the functional specificity of rab5. To test the role of these elements experimentally, we transplanted them onto rab6, which is associated with the Golgi complex. The chimeric proteins were assayed for intracellular localization and stimulation of endocytosis. First, we found that the C-terminus of rab5 could target rab6 to the plasma membrane and early endosomes but it did not confer rab5-like stimulation of endocytosis. Further replacement of other regions revealed that the N-terminus, helix alpha 2/loop 5 and helix alpha 2/loop 7 were all required to functionally convert rab6 into rab5. Reciprocal hybrids of rab5 containing these regions replaced with those of rab6 were inactive, demonstrating that each region is essential for rab5 function. These results indicate that distinct structural elements specify the localization, membrane association and regulatory function of rab5.

Amino Acid Sequence↗
PubMed1994

Isolation of a mouse cDNA encoding Rab23, a small novel GTPase expressed predominantly in the brain.

The full-length cDNA encoding Rab23, a novel Ras-related small GTPase, was isolated using the sequence of a previously described [Chavrier et al., Gene 112 (1992) 261-264] short cDNA fragment and the rapid amplification of cDNA ends (RACE) PCR techniques. The deduced amino acid sequence was not very closely related to any previously described small GTPase, but was within the Rab subfamily. A Northern analysis revealed that the rab23 mRNA is predominantly expressed in the brain, which places the protein, together with Rab3a and Rab15, in the group of small GTPases characteristic of the nervous system.

Amino Acid Sequence↗
PubMed1994

The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis.

Rab5a is a small GTPase that regulates fusion of endocytic vesicles to early endosomes. We investigated whether Rab5a is involved in early endocytic traffic in both the axonal and the somatodendritic domains of polarized neurons. Using immunofluorescence, endogenous Rab5a was detected in axons and dendrites. Its localization in axons strongly overlapped that of the synaptic vesicle protein synaptophysin. Indeed, Rab5a co-immunoisolated with synaptophysin-containing vesicles, and antibodies against Rab5a labeled synaptic vesicle-like structures in nerve terminals. The functional association of Rab5a with dendritic and axonal early endosomes was assayed by electron microscopy after overexpression of wild-type and mutant Rab5a in cultured hippocampal neurons. This induced the formation of abnormal endosomes in both the somatodendritic and the axonal domains. These results show a role for Rab5a in axonal and dendritic endocytosis, and the presence of Rab5a on synaptic vesicles indicates that the axonal endosomes participate in the biogenesis of these vesicles.

Animals↗
PubMed1994

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast.

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.

Amino Acid Sequence↗
PubMed1994

Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression.

Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell type-dependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.

Amino Acid Sequence↗
PubMed1994

Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex.

Among the small GTPases of the Ras family, Rap proteins exhibit the highest homology with p21Ras. The four Rap proteins so far identified constitute two subgroups, comprising the Rap1(A,B) and the Rap2(A,B) proteins. The intracellular location of Rap1A, Rap1B and Rap2A proteins was investigated in mammalian cells by confocal immunofluorescence microscopy. Using a specific anti-Rap1 affinity-purified antibody, both Rap1A and Rap1B proteins were localized to late endocytic compartments (late endosomes/lysosomes) in fibroblasts. The localization of the Rap1A and B proteins transiently overexpressed with the vaccinia T7 system was identical to that observed for endogenous Rap1 proteins. In contrast, epitope-tagged Rap2A protein colocalized with several markers of the Golgi complex, thus indicating that its site of function was distinct from that of Rap1A. In addition, morphological and subcellular fractionation studies provided evidence for the association of Rap1 proteins with phagosomes displaying biochemical features of late endocytic structures in J774 macrophages. Thus, the localization of Rap1A and Rap1B implicates their involvement in late endocytic/phagocytic processes.

Animals↗
PubMed1993

Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells.

A specific polyclonal antibody was used to investigate the subcellular distribution of the small GTPase, rab11p, in the neuroendocrine cell line, PC12. We took advantage of a previously described pulse-chase protocol based on sulfation to examine the distribution of rab11 along the secretory pathway. Using the rab11 antiserum, but not serum depleted of rab11 antibodies, we were able to specifically immunoisolate markers of the constitutive and the regulated secretory pathway in the trans-Golgi network (TGN) as well as after their exit from this compartment (constitutive secretory vesicles, immature, and mature secretory granules). We therefore conclude that rab11p is associated with the TGN and with TGN-derived vesicles of both the constitutive and the regulated secretory pathway in PC12 cells.

Animals↗
PubMed1993

Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins.

Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic. These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles. Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane. In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins. Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins. However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif. Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways. Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI.

Amino Acid Sequence↗
PubMed1993

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.

Amino Acid Sequence↗
PubMed1993

Rab GTPases in vesicular transport.

Specificity and directionality are two features shared by the numerous steps of membrane transport that connect cellular organelles. By shuttling between specific membrane compartments and the cytoplasm, small GTPases of the Rab family appear to regulate membrane traffic in a cyclical manner. The restriction of certain Rab proteins to differentiated cell types supports a role for these GTPases in defining the specificity of membrane trafficking.

Animals↗
PubMed1993

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization.

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell-specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.

Amino Acid Sequence↗
PubMed1993

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane.

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.

ADP-Ribosylation Factors↗
PubMed1993

Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p.

In the companion paper (Huber, L. A., S. W. Pimplikar, R. G. Parton, H. Virta, M. Zerial, and K. Simons. J. Cell Biol. 123:35-45) we reported that the small GTPase rab8p is involved in transport from the TGN to the basolateral plasma membrane in epithelia. In the present work we investigated the localization and function of rab8p in polarized hippocampal neurons. By immunofluorescence microscopy we found that rab8p localized preferentially in the somatodendritic domain, and was excluded from the axon. Double-labeling immunofluorescence showed that some of the rab8p co-localized in the dendrites with the Semliki Forest Virus glycoprotein E2 (SFV-E2). An antisense oligonucleotide approach was used to investigate the role of rab8p in dendritic transport of newly synthesized viral glycoproteins. Antisense oligonucleotides corresponding to the initiation region of the rab8 coding sequence were added to the cultured neurons for four days. This treatment resulted in a significant decrease in cellular levels of rab8p and transport of SFV-E2 from the cell body to the dendrites was significantly reduced. However, no effect was observed on axonal transport of influenza HA. From these results we conclude that rab8p is involved in transport of proteins to the dendritic surface in neurons.

Animals↗
PubMed1993

Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily.

Small GTPases of the rab subfamily are involved in regulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have not been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was localized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endosomal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.

Amino Acid Sequence↗
PubMed1993

Regulation of endocytosis by the small GTP-ase rab5.

Rab5 is a small GTPase associated with the plasma membrane and the early endosomes. Expression of wild type rab5 and a mutant protein defective in GTP-binding in BHK cells led to alterations in the rate of endocytosis and in the morphology of endocytic organelles. The results obtained suggest that rab5 is a rate limiting GTPase that regulate the kinetics of both lateral fusion of early endosomes and fusion of plasma membrane-derived endocytic vesicles with early endosomes.

Animals↗