FYVE Domain Targets Pib1p Ubiquitin Ligase to Endosome and Vacuolar Membranes

Abstract

Signaling by phosphatidylinositol 3-kinases (PI3Ks) is often mediated by proteins which bind PI3K products directly and are localized to intracellular membranes rich in PI3K products. The FYVE finger domain binds with high specificity to PtdIns(3)P and proteins containing this domain have been shown to be important components of diverse PI3K signaling pathways. The genome of the yeast Saccharomyces cerevisiae encodes five proteins containing FYVE domains, including Pib1p, whose function is unknown. In addition to a FYVE finger motif, the primary structure of Pib1p contains a region rich in cysteine and histidine residues that we demonstrate binds 2 mol eq of zinc, consistent with this region containing a RING structural domain. The Pib1p RING domain exhibited E2-dependent ubiquitin ligase activity in vitro, indicating that Pib1p is an E3 RINGtype ubiquitin ligase. Fluorescence microscopy was used to demonstrate that a GFP-Pib1p fusion protein localized to endosomal and vacuolar membranes and deletional analysis of Pib1p domains indicated that localization of GFP-Pib1p is mediated solely by the FYVE domain. These results suggest that Pib1p mediates ubiquitination of a subset of cellular proteins localized to endosome and vacuolar membranes, and they expand the repertoire of PI3K-regulated pathways identified in eukaryotic cells.The genome of the budding yeast, Saccharomyces cerevisiae, encodes a single phosphatidylinositol 3-kinase (PI3K), 1 encoded by the VPS34 gene, that is responsible for all PtdIns(3)P synthesis within the cell (1). The only known function of the yeast Vps34 PI3K is in sorting of proteins to the lysosome-like vacuole via multiple pathways (1-3). Vps34p directly regulates Golgi-to-endosome vesicle-mediated transport via the PtdIns(3)P-binding protein, Vac1/Pep7p (4 -8), required for docking and fusion of Golgi-derived vesicles with the prevacuolar endosome. Vac1p, and the related human protein EEA1, link PI3K signaling directly to SNARE-mediated vesicle trafficking within endosomal pathways by directly binding PtdIns(3)P, SNAREs, and other required factors such as the SEC1 family protein, Vps45p (8,9). In addition to this role in regulating fusion of post-Golgi membranes, Vps34p produces substrate for the Fab1 phosphatidylinositol 3-phosphate 5-kinase, which has been implicated in protein sorting within late endosomes and multivesicular body endosomes (10). Besides these roles for PtdIns(3)P, other roles are likely to be discovered as more PtdIns(3)P-binding proteins are characterized.The downstream effects of PI3K signaling are, in many cases, mediated by proteins containing a PtdIns(3)P-binding protein module called a FYVE finger domain. FYVE domains comprise a subfamily of RING domains, identified by the spacing of cysteine and/or histidine residues which bind two atoms of zinc and are required for the overall structure of the domain (11-13). The hallmark of the FYVE finger subfamily is a patch of basic residues surrounding the third zinc-coordinating cysteine residue which forms, together ...

hRUL138, a novel human RNA-binding RING-H2 ubiquitin-protein ligase

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Modulation of Ubc4p/Ubc5p-Mediated Stress Responses by the RING-Finger-Dependent Ubiquitin-Protein Ligase Not4p in Saccharomyces cerevisiae

“…For instance, Pib1p has been described to contain RING-finger-dependent E3 ligase activity in vitro (Shin et al 2001). Possibly, Pib1p constitutes a redundant E3 ligase for Not4p under certain conditions.…”

Phosphoinositides in Constitutive Membrane Traffic

“…Clathrin binding enhanced the affinity of AP2 for internalization signals to a similar extent, but the effect of clathrin and PtdIns(3)P were not additive (286). However, when fluorescent proteins that bind PtdIns(3)P are expressed in cells, they label endosomes and not the plasma membrane (32,92,318), suggesting that there is little free PtdIns(3)P at the cell surface. There is a recent report that PtdIns3KIIC2␣ localizes to the nucleus rather than the plasma membrane (77).…”

“…At higher expression levels, they will interfere with the normal regulation of PIP production. With these limitations in mind, PtdIns(3)P has been detected primarily on endocytic membranes (32,92,318), PtdIns(4,5)P 2 at the plasma membrane (148,149,219,234,365) and on internal membranes enriched in lipid rafts (27,297), and PtdIns(4)P on the Golgi (13,201,372). PtdIns4K activity has been detected in cell fractions enriched in lysosomes (7,54,56).…”