Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4

Rabip4 is a Rab4 effector, which possesses a RUN domain, two coiled-coil do mains, and a FYVE finger. It is associated with the early endosomes and lea ds, in concert with Rab4, to the enlargement of endosomes, resulting in the fusion of sorting and recycling endosomes. Our goal was to characterize th e role of these various domains in Rabip4 subcellular localization and thei r function in Chinese hamster ovary cells. Although the FYVE finger domain specifically bound phosphatidylinositol 3-phosphate and was necessary for t he function of Rabip4, it was not sufficient for the protein association wi th membranes. Indeed a protein containing the FYVE finger and the Rab4-bind ing site was cytosolic, whereas the total protein was mostly associated to the membrane fraction, whether or not cells were pretreated with wortmannin . By contrast, a construct corresponding to the N-terminal end, Rabip4-(1-2 12), and containing the RUN domain was membrane-associated. The complete pr otein partitioned between the Triton X-100-insoluble and -soluble fractions and a wortmannin treatment increased the amount of the protein in the Trit on X-100 fraction. Rabip4-(1-212) was totally Triton X-100-insoluble, and c onfocal microscopic examination showed that it labeled not only the endosom es, positive for Rabip4, but also a filamentous network with a honeycomb ap pearance. The Triton X-100-insoluble fraction that contains Rabip4 did not correspond to the caveolin or glycosylphosphatidylinositol-enriched lipid r afts. Rabip4 did not appear directly linked to actin but seemed associated to the actin network. We propose that the subcellular localization of the p rotein is primarily driven by the RUN domain to endosomal microdomains char acterized by Triton X-100 insolubility and that the FYVE domain and the Rab 4-binding domain then allow for the recruitment of the protein to lipophili c microdomains enriched in phosphatidylinositol 3-phosphate.