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      Distribution of Rab GTPases in Mouse Kidney and Comparison with Vacuolar H +-ATPase

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          Background: Vacuolar H<sup>+</sup>-ATPases (V-ATPases) are essential for renal bicarbonate transport in both the proximal and distal nephron. Regulation of proton transport occurs, in part, by vesicle-mediated traffic of V-ATPases between intracellular vacuoles and the plasma membrane. Although the proteins involved in regulated V-ATPase traffic are largely unknown, Rab GTPases have a central role in the traffic and recycling of other membrane proteins. Methods: To identify candidate Rab GTPases potentially involved in V-ATPase traffic, immunocytochemical and subcellular fractionation studies were used to evaluate the distribution to sites of abundant V-ATPase of 5 Rab GTPases expressed in kidney, Rab5a, Rab11, Rab13, Rab18, and Rab20. Results: The immunocytochemical distribution of Rab5a and Rab13 and the subcellular distribution of Rab18 were not compatible with a role in V-ATPase traffic. In contrast, Rab11 colocalized with V-ATPase in apical regions of proximal tubule, and Rab20 colocalized with the enzyme in intercalated cells. Rab11 and Rab20 were enriched in membrane fractions that were also enriched in V-ATPase B2 and B1 subunit isoforms, respectively. Conclusions: The immunohistochemical data in combination with the membrane fractionation studies are consistent with a potential role for Rab11 and Rab20 in regulating V-ATPase traffic in specific segments of the nephron.

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          Most cited references 16

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          Osteoclastic bone resorption by a polarized vacuolar proton pump.

          Bone resorption depends on the formation, by osteoclasts, of an acidic extracellular compartment wherein matrix is degraded. The mechanism by which osteoclasts transport protons into that resorptive microenvironment was identified by means of adenosine triphosphate-dependent weak base accumulation in isolated osteoclast membrane vesicles, which exhibited substrate and inhibition properties characteristic of the vacuolar, electrogenic H+-transporting adenosine triphosphatase (H+-ATPase). Identify of the proton pump was confirmed by immunoblot of osteoclast membrane proteins probed with antibody to vacuolar H+-ATPase isolated from bovine kidney. The osteoclast's H+-ATPase was immunocytochemically localized to the cell-bone attachment site. Immunoelectron microscopy showed that the H+-ATPase was present in the ruffled membrane, the resorptive organ of the cell.
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            Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

            Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.
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              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.

                Author and article information

                Nephron Physiol
                Nephron Physiology
                S. Karger AG
                July 2005
                10 June 2005
                : 100
                : 3
                : p31-p42
                Departments of aMedicine and bAnatomy and Cell Biology, University of Florida, Gainesville, Fla., USA
                85114 Nephron Physiol 2005;100:p31–p42
                © 2005 S. Karger AG, Basel

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                Page count
                Figures: 7, Tables: 2, References: 39, Pages: 1
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/85114
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