A novel role for Rab5–GDI in ligand sequestration into clathrin-coated pits

McLauchlan, Hilary; Newell, J. Glen; Morrice, Nick; Osborne, Andrew R.; West, Michele; Smythe, Elizabeth

doi:10.1016/s0960-9822(98)70018-1

Cited by 254 publications

(204 citation statements)

References 53 publications

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“…The polyphosphoinositidebinding site of the ␣ subunit of AP-2 is required for targeting to clathrin-coated pits (Gaidarov and Keen, 1999), implying that lipid kinases could modulate adaptor recruitment. Consistently, adaptor-dependent, de novo-coated pit formation requires ATP, although it is presently unknown whether this reflect lipid or protein modification (McLauchlan et al, 1997). Cytosolic AP-2 is phosphorylated, whereas AP-2 on membranes is not (Wilde and Brodsky, 1996), implying that AP-2 dephosphorylation could be necessary for recruitment to its donor compartment.…”

Section: Ap-3 Adaptor Phosphorylation By Casein Kinasementioning

confidence: 99%

The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

Faúndez

Kelly

2000

MBoC

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The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes . When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the ␤3 subunit of the complex by a kinase similar to casein kinase 1␣. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the ␤3A and ␤3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes. INTRODUCTIONMembrane proteins are carried from donor membranes to acceptor membranes by two steps, the vesiculation of a carrier vesicle from the donor and the fusion of the carrier vesicle with the acceptor. Vesiculation of the donor membrane can itself be divided into five steps: recognition of cargo proteins; recruitment of coating molecules; the imposition of curvature on the forming vesicle membrane; fission of the carrier vesicle from the donor membrane; and, finally, the loss of the vesicle coat (Springer et al., 1999). To discover the molecular events that take place at each step, several laboratories have reconstituted either the entire process of vesiculation or individual steps in vitro.The first two steps of vesiculation, cargo recognition and coating, can be regulated by phosphorylation. Membrane proteins are recognized as cargo because they contain sorting domains. Adaptors interact with a tyrosine or a dileucine motif (Schmid, 1997;Kirchhausen et al., 1997;Bonifacino and Dell'Angelica, 1999). The recognition of cargo molecules can be regulated by the phosphorylation of sites close to the sorting domains; for example, in the trafficking of furin (Wan et al., 1998;Teuchert et al., 1999;Molloy et al., 1999), CD4 (Pitcher et al., 1999), CTLA-4 (Shiratori et al., 1997), MHC-II-Ii complex (Anderson and Roche, 1998), and pIgAR (Casanova et al., 1990;Okamoto et al., 1994;Luton et al., 1998). The cargo molecules are recruited into a newly forming carrier vesicle by cytoplasmic coat molecules (Matsuoka et al., 1998;Bremser et al., 1999). The recruitment of two of the known coats, COPI and COPII, is regulated by small ARF-like GTPases and appears to be a relatively simple p...

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Section: Ap-3 Adaptor Phosphorylation By Casein Kinasementioning

confidence: 99%

The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

Faúndez

Kelly

2000

MBoC

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“…Although rab proteins have been primarily associated with vesicletargeting functions, there are a number of examples in which the rab proteins have been suggested to influence vesicle budding (Plutner et al, 1991;Riederer et al, 1994;Jedd et al, 1995Jedd et al, , 1997. Perhaps the most compelling evidence derives from in vitro assays showing a direct role for rab5 in coated vesicle formation at the plasma membrane (McLauchlan et al, 1998). Rab5 has also been demonstrated to control the fusion of clathrin-coated vesicles with endosomes as well as the homotypic fusion of early endosomes (Gorvel et al, 1991).…”

Section: Rab11 Function In Constitutive Exocytosismentioning

confidence: 99%

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

Chen

Feng

Chen

et al. 1998

MBoC

350

293

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The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum–to–Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

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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%

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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A novel role for Rab5–GDI in ligand sequestration into clathrin-coated pits

Cited by 254 publications

References 53 publications

The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

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

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