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 determi ne the mechanism underlying this change in distribution, me establishe d an in vitro assay that reconstituted specific binding of rab4 when e ndosome-containing membranes were incubated with rab4 complexed with i ts cytosolic chaperone, GDP dissociation inhibitor (GDI), rab4 was fou nd to bind to a saturable receptor associated with highly purified end osomes, Membrane binding and nucleotide exchange were physically disti nct, since an active soluble fragment of the rab4 receptor, but not ra b4 nucleotide exchange activity, could be released from membranes by e lastase 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 com plexes, but did completely inhibit rab4 binding to its receptor, In co ntrast, in vitro phosphorylation of membranes did not result in the di ssociation of bound rab4, nor were mitotic membranes deficient with re spect to binding non-phosphorylated rab4. Thus, mitotic cells appear t o accumulate rab4 in the cytosol by phosphorylating rab4 during the so luble phase of its normal activity cycle, thereby preventing membrane attachment.