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M Zerial

Publications and source records attributed to M Zerial.

At least 37 records · Page 2Linked to original sources
PubMed1999

Rab5 regulates motility of early endosomes on microtubules.

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.

Biological Transport↗
PubMed1999

Visualization of receptor-mediated endocytosis in yeast.

We studied the ligand-induced endocytosis of the yeast alpha-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to alpha-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Delta. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.

Actins↗
PubMed1998

EEA1 links PI(3)K function to Rab5 regulation of endosome fusion.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Androstadienes↗
PubMed1998

Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion.

Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Amino Acid Sequence↗
PubMed1998

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

Amino Acid Sequence↗
PubMed1998

Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

Amino Acid Sequence↗
PubMed1998

HMG-17, a chromosomal non-histone protein, shows developmental regulation during organogenesis.

We used the differential hybridization technique for isolating developmentally regulated genes from the mouse metanephric kidney. In this screening, we identified the cDNA encoding high-mobility-group protein 17 (HMG-17), a chromosomal non-histone protein which modulates the conformation of transcriptionally active chromatin. Using Northern blot analysis, the HMG-17 mRNA was strongly expressed during embryogenesis and downregulated in various adult murine organs. At the histological level, the transcript localized to differentiating tissue regions and was apparently downregulated in mature structures indicating that HMG-17 expression is linked to cell differentiation. HMG-17 can thus be regarded as a general marker for tissues or cells undergoing differentiation during organogenesis.

Animals↗
PubMed1997

Rab17 and rab18, small GTPases with specificity for polarized epithelial cells: genetic mapping in the mouse.

The Rab subfamily of small GTPases plays an important role in the regulation of membrane traffic in eukaryotic cells. While most Rab proteins are equally expressed in polarized and nonpolarized cells, Rab17 and Rab18 show epithelial cell specificity. Here we report the genetic mapping of Rab17 and Rab18 on mouse chromosomes 1 and 18, respectively. We also discuss some implications of Rab17 and Rab18 mapping, including their candidacy for the mouse mutations ln (leaden), Tw (twirler), and ax (ataxia).

Animals↗
PubMed1997

Cleavage of rabaptin-5 blocks endosome fusion during apoptosis.

Cells undergoing apoptosis exhibit striking changes in membrane organization, including plasma membrane blebbing and invagination, vacuolation and fragmentation of organelles, and alterations in the surface expression of receptors. The underlying mechanisms for these changes are unknown, though alterations in vesicular fusion are likely to play a role. Using a cell-free system based on Xenopus laevis egg extracts we have found that endosome fusion is blocked during apoptosis. Inhibition of fusion is prevented by Bcl-2 or Bcl-xL, two negative regulators of apoptosis, or by specific inhibitors of members of the caspase family of apoptotic proteases. Selective cleavage of Rabaptin-5, an essential and rate-limiting component of endosome fusion, is responsible for the loss of fusion activity. Cleavage of Rabaptin-5 also occurs in cellular models for apoptosis. These results suggest that inactivation of Rabaptin-5 and inhibition of vesicle transport lead to fragmentation of endosomes and inhibition of the endocytic pathway during the execution phase of apoptosis. We propose that parallel changes to other membrane transport pathways would give rise to general membrane fragmentation in apoptotic cells. These changes are likely to play an important role in the generation of apoptotic bodies and their recognition by phagocytosing cells.

Animals↗
PubMed1997

A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function.

The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.

Animals↗
PubMed1997

The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin.

The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant-negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild-type or GTPase-deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth.

Bacterial Proteins↗
PubMed1997

Rab7: NMR and kinetics analysis of intact and C-terminal truncated constructs.

Rab proteins are a family of approximately 25kD ras-related GTPases which are associated with distinct intracellular membranes where they control vesicle traffic between intracellular compartments. The late-endosomal rab protein rab7(1-207), (lacking only the C-terminal lipids of the native molecule) and three C-terminal truncated constructs rab7(1-202), rab7(1-197) and rab7(1-182) were purified using an E. coli expression system. The C-terminal tail region of rab proteins is of special interest because it is thought to target rab proteins to particular intracellular membranes. A comparison of TOCSY-NMR spectra from intact rab7(1-207) and the tail-less construct rab7(1-182) suggested that much of the C-terminal tail is flexible in solution. The GTP hydrolysis, and GDP association and dissociation rates for all the truncated and intact constructs were similar, showing that the tail region of rab7(1-207) has little influence on the hydrolysis and exchange rates of the nucleotide.

GTP-Binding Proteins↗
PubMed1997

The diversity of Rab proteins in vesicle transport.

Rab proteins have been primarily implicated in vesicle docking as regulators of SNARE pairing. Recent findings, however, indicate that their function in vesicle trafficking can go beyond this role, and a number of proteins, unrelated to each other, have been identified as putative Rab effectors. Although the GTPase switch of Rab proteins is highly conserved, functional mechanisms may be highly diversified among members of the Rab family.

Amino Acid Sequence↗
PubMed1997

HSV infection of polarized epithelial cells on filter supports: implications for transport assays and protein localization.

Epithelial cell lines can be grown on filter supports and form polarized monolayers with distinct basolateral and apical plasma membrane domains. This property has been extensively used in cell biology to investigate epithelial cell function. To date, a major limitation of this approach has been the difficulty of obtaining transient gene expression in polarized epithelia. Here we present an approach to overcome this problem using gene transfer into polarized epithelial cells grown on filters using a herpes virus-based vector. Recombinant genes are inserted into a defective HSV-1 plasmid and packaged with a replication-incompetent HSV-1 helper virus into virus particles which are used to infect the polarized epithelial cells grown on filters. The transepithelial resistance of the cells is not affected by the addition of virus, and there are no detectable cytopathic effects.

Animals↗
PubMed1996

Endosome dynamics regulated by a Rho protein.

Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.

Actins↗
PubMed1996

GTPase activity of Rab5 acts as a timer for endocytic membrane fusion.

The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport. Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein. To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5. Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.

ADP-Ribosylation Factors↗