Mitotic phosphorylation of rab4 prevents binding to a specific receptor on endosome membranes.
Phosphorylation of the monomeric GTPase rab4 in mitotic cells leads to its relocalization from endosome membranes to the cytosol. To determine the mechanism underlying this change in distribution, we established an in vitro assay that reconstituted specific binding of rab4 when endosome-containing membranes were incubated with rab4 complexed with its cytosolic chaperone, GDP dissociation inhibitor (GDI). rab4 was found to bind to a saturable receptor associated with highly purified endosomes. Membrane binding and nucleotide exchange were physically distinct, since an active soluble fragment of the rab4 receptor, but not rab4 nucleotide exchange activity, could be released from membranes by elastase cleavage. Interestingly, the soluble fragment could be used to fully reconstitute rab4 membrane binding. In vitro phosphorylation of rab4 by cdc2/cyclin B kinase did not affect formation of rab4-GDI complexes, but did completely inhibit rab4 binding to its receptor. In contrast, in vitro phosphorylation of membranes did not result in the dissociation of bound rab4, nor were mitotic membranes deficient with respect to binding non-phosphorylated rab4. Thus, mitotic cells appear to accumulate rab4 in the cytosol by phosphorylating rab4 during the soluble phase of its normal activity cycle, thereby preventing membrane attachment.
Ayad
N
N
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520-8002, USA.
Hull
M
M
Mellman
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I
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008938:Mitosis; D010196:Pancreatic Elastase / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012995:Solubility; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins
C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D016203:CDC2 Protein Kinase; D010196:Pancreatic Elastase; D019204:GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins