Capture of the small GTPase Rab5 by GDI: regulation by p38 MAP kinase.
The small GTPase Rab5 is one of the key regulators of early endocytic traffic and, like other GTPases, cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle is controlled by a guanine nucleotide dissociation inhibitor (GDI), which functions as a Rab vehicle in the cytosol. GDI extracts from membranes the inactive GDP-bound form of the Rab. Then, the cytosolic GDI:Rab complex is delivered to the appropriate target membrane, where the Rab protein is reloaded, presumably via a GDI displacement factor (Pfeffer and Aivazian, 2004). We previously reported that the formation of the GDI:Rab5 complex is stimulated by the mitogen-activated protein kinase p38 (Cavalli et al., 2001). Mol. Cell7, 421-432.]. Selective activation of p38 MAPK increases endocytic rates in vivo, presumably allowing more efficient internalization of cell surface components for repair, storage, or degradation. These observations emphasize the possibility that external stimuli contribute to the regulation of membrane traffic. Here, we describe how to monitor the ability of GDI to extract Rab5 from early endosomal membranes in vitro and the role of p38 MAPK in this process. In addition, we detail how to investigate the possible role of p38 MAPK in the regulation of endocytosis in vivo.
Michela
M
Felberbaum-Corti
Valeria
V
Cavalli
Jean
J
Gruenberg
D001483:Base Sequence; D017931:DNA Primers; D004705:Endocytosis; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000502:physiology*; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000378:metabolism; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000502:physiology*
D017931:DNA Primers; D020730:Guanine Nucleotide Dissociation Inhibitors; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D048051:p38 Mitogen-Activated Protein Kinases; D020696:rab5 GTP-Binding Proteins