rab5 controls early endosome fusion in vitro

Gorvel, Jean‐Pierre; Chavrier, Philippe; Zerial, Marino; Grüenberg, Jean

doi:10.1016/0092-8674(91)90316-q

Cited by 1,008 publications

(931 citation statements)

References 48 publications

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“…3A). We also prepared an early-endosome-enriched fraction from BHK cells with a well-established flotation gradient as described by Gorvel et al (9). Again, Hrs association with this fraction was wortmannin sensitive (Fig.…”

Section: Resultsmentioning

confidence: 99%

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Urbé

Mills

Stenmark

et al. 2000

Molecular and Cellular Biology

117

120

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Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a prominent target for tyrosine phosphorylation following the activation of tyrosine kinase receptors (11). It was initially shown to lie downstream of the HGF (scatter factor) receptor c-met, but activation of other tyrosine kinase receptors and by cytokines such as interleukin-2 and granulocyte-macrophage colony-stimulating factor also results in phosphorylation of Hrs (1). It is localized to transferrin receptor-containing endosomes (12) and bears significant similarity (including a FYVE-finger motif and a VHS domain) to the Saccharomyces cerevisiae protein Vps27. Vps27 belongs to the class E set of Vps mutants which are defective in transport from the sorting endosome to the vacuole (2, 22). A highly related protein, Hrs-2, has been shown to interact with SNAP-25 and SNAP-23, homologous proteins which are involved in regulated exocytosis and other intracellular fusion events, respectively (3).Two proteins that bind to Hrs have been identified which have been named STAM (for signal-transducing adapter molecule) and Hrs binding protein (Hbp) (1, 31). They show 53% sequence identity, each bears an SH3 domain, and both are also tyrosine phosphorylated. In T cells, overexpression of Hrs leads to suppression of cytokine-mediated DNA synthesis, while a mutant unable to bind STAM is without effect (1). NIH 3T3 cells stably transfected with mutants of Hbp that lack the SH3 domain or the binding site for Hrs are impaired in degrading internalized platelet-derived growth factor (31). This latter result is consistent with a role for Hrs in regulating transport from early to late endosomes (or multivesicular bodies) proposed by analogy to Vps27 function in yeast.The FYVE domain is a double zinc finger domain that has been shown ...

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Section: Resultsmentioning

confidence: 99%

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Urbé

Mills

Stenmark

et al. 2000

Molecular and Cellular Biology

117

120

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“…mutants. The biological function of Rab5 is to regulate endocytosis by promoting early endosome fusion [31,35,36], and possibly vesicle budding at the plasma membrane [37]. As a result, overexpression of Rab5 in cultured cells increased the endocytic activity by 2-fold, as determined by HRP uptake [23,24].…”

Section: Figure 2 Single-step Gtp Hydrolysis By the Gst Fusion Proteimentioning

confidence: 99%

“…The biological function of Rab5 is to promote early endosome fusion [31,35,36]. A recent study also suggests the involvement of Rab5 in vesicle budding from the plasma membrane [37].…”

Section: Figure 4 Molecular Modelling Of An Arg 13 Substitution (Equimentioning

confidence: 99%

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

Liang

Mather

2000

Biochemical Journal

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Structural and biochemical data indicate the importance of the phosphate-binding loop residues Gly"# and Gly"$ of Ras both in the GTP hydrolysis reaction and in biological activity, but these two residues are not conserved in other Ras-related GTPases. To gain a better understanding of this region in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPase as a model for comparison, and substituted the Ala$! residue (the equivalent of Gly"$ of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp contrast with the observation that a proline substitution at the neighbouring position (Gly"# of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution

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“…To establish its position unambigously, rab5 was overexpressed using the T7 RNA polymerase recombinant vaccinia virus system (Chavrier et al, 1990a;Gorvel et al, 1991) and early endosomal membranes were prepared using the flotation gradient. These fractions were then treated with trypsin as above [1:1000 (w/w) trypsin:endosomal protein], recovered by centrifugation and analyzed by two-dimensional gel electrophoresis.…”

Section: Rab5 Associated With Early Endosomal Membranes Is Trypsin-sementioning

confidence: 99%

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

et al. 1994

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Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.

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rab5 controls early endosome fusion in vitro

Cited by 1,008 publications

References 48 publications

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

Endosomal Localization and Receptor Dynamics Determine Tyrosine Phosphorylation of Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

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