The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway

Bucci, Cecilia; Parton, Robert G.; Mather, Ian H.; Stunnenberg, Henk; Simons, Kai; Hoflack, Bernard; Zerial, Marino

doi:10.1016/0092-8674(92)90306-w

Cited by 1,290 publications

(994 citation statements)

References 60 publications

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“…A recent study also suggests the involvement of Rab5 in vesicle budding from the plasma membrane [37]. As a result, overexpression of Rab5 in cultured cells stimulates both receptor-mediated and fluid-phase endocytosis [23,24]. In the present study, we have shown that the GTPhydrolysis-defective Pro$!…”

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

confidence: 65%

“…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]. This stimulation of HRP uptake was used as a biological measure of Rab5 activity.…”

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

confidence: 99%

“…To investigate whether the position of Gly"$ (backbone amide and side chain) is important in GTP hydrolysis and biological function of other GTPases, we used Rab5, which is a member of the Ras-related GTPase family and a regulator of endocytosis [23,24], as a model GTPase, and substituted Ala$! of Rab5 (the equivalent of Gly"$ in Ras) with all the other 19 amino acids.…”

Section: Introductionmentioning

confidence: 99%

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

confidence: 65%

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

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…This conclusion is further strengthened by our demonstration that the distribution of rab5 is markedly changed on sucrose gradients. We have shown that in control cells, rab5, a marker for early endosomes [34,41], exhibits a similar distribution to GLUT4 following sucrose-density-gradient analysis of intracellular membranes (Figure 8). However, after ablation, rab5 but not GLUT4 shifts into a more dense region of the gradient.…”

Section: Okadaic Acid On Deglc Transport and Glut4 Ablation In 3t3-l1mentioning

confidence: 73%

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

Livingstone

James

Rice

et al. 1996

Biochemical Journal

138

159

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The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained 90 % of the immunoreactive GLUT4 found in this fraction, but only 40 % of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble (' ablate ') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 mC resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40 % of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant

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“…We have moreover made MDCK cell lines stably expressing a dominant negative mutant of Rab5 or transiently expressing Rab GDI, and have shown that the Rho and Rab families coordinately regulate cell adhesion and migration (Imamura et al, 1998). The Rab small G protein family is implicated in intracellular vesicle tra cking and Rab5 is involved in endocytosis (Bucci et al, 1992;Stenmark et al, 1994;for review, see Novick and Zerial, 1997). Rab GDI is a general regulator of all the Rab family members (for reviews, see Takai et al, 1996;Novick and Zerial;.…”

Section: Introductionmentioning

confidence: 99%

Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells

et al. 1999

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The Rho small G protein family consists of the Rho, Rac, and Cdc42 subfamilies and regulates various cell functions through reorganization of the actin cytoskeleton. We previously showed that the Rho subfamily regulates the formation of stress ®bers and focal adhesions whereas the Rac subfamily regulates the Ecadherin-based cell-cell adhesion in MDCK cells. We studied here the function of the Cdc42 subfamily, consisting of two members, Cdc42Hs and G25k, in cell adhesion, migration, and morphology of MDCK cells. For this purpose, we made and used MDCK cell lines stably expressing each of dominant active mutants of Cdc42Hs (sMDCK-Cdc42HsDA) and G25K (sMDCK-G25KDA). Actin ®laments at the cell-cell adhesion sites increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Both E-cadherin and b-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in both sMDCK-Cdc42HsDA and -G25KDA cells. Electron microscopic analysis revealed that sMDCKCdc42HsDA cells tightly contacted with each other throughout the lateral membranes. Moreover, both the HGF-and TPA-induced disruption of the cadherin-based cell-cell adhesion and the subsequent cell migration were inhibited in both sMDCK-Cdc42HsDA and -G25KDA cells. Co-expression of the dominant negative mutant of Rac1, a member of the Rac subfamily, with the dominant active mutant of Cdc42Hs did not inhibit the increased accumulation of actin ®laments at the cell-cell adhesion sites. These results suggest that the Cdc42 subfamily is involved in the cadherin-based cell-cell adhesion in a manner independent of the Rac subfamily. Furthermore, the cells were frequently enveloped by the large multinuclear cells in both sMDCK-Cdc42HsDA and -G25KDA cells. Video microscopic analysis revealed that the cells were engulfed by the large cells during cytokinesis.

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The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway

Cited by 1,290 publications

References 60 publications

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

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

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells

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