FYVE domain targets Pib1p ubiquitin ligase to endosome and vacuolar membranes.
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 PtdIns3P 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 RING-type 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.
Shin
M E
ME
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, USA.
Ogburn
K D
KD
Varban
O A
OA
Gilbert
P M
PM
Burd
C G
CG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D011992:Endosomes / Q000201:enzymology*; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000201:enzymology; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014617:Vacuoles / Q000201:enzymology*
D049452:Green Fluorescent Proteins; D008025:Ligases; D008164:Luminescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; C469288:PIB1 protein, S cerevisiae