A Novel Membrane-anchored Rab5 Interacting Protein Required for Homotypic Endosome Fusion
| Autor: | P. D. Stahl, S. Hoffenberg, Wenping Dai, H. S. Hall, L. Nikolova, Brian J. Knoll, M. A. Barbieri, R. E. Baughn, Burton F. Dickey, X. Liu, Alejandro Aballay |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Sindbis virus
Endosome Recombinant Fusion Proteins Green Fluorescent Proteins Molecular Sequence Data Mutant Protein Prenylation Endosomes Saccharomyces cerevisiae GTPase Transfection Guanosine Diphosphate Membrane Fusion Biochemistry Cell Line Cricetinae Animals Humans Amino Acid Sequence Molecular Biology rab5 GTP-Binding Proteins Sequence Homology Amino Acid biology fungi Intracellular Signaling Peptides and Proteins Brain Membrane Proteins Intracellular Membranes Cell Biology Thionucleotides biology.organism_classification Cell biology Molecular Weight Kinetics Luminescent Proteins Cytosol Transmembrane domain Endocytic vesicle Guanosine 5'-O-(3-Thiotriphosphate) Mutagenesis Site-Directed biological phenomena cell phenomena and immunity Carrier Proteins Sequence Alignment HeLa Cells |
| Zdroj: | Journal of Biological Chemistry. 275:24661-24669 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m909600199 |
| Popis: | The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5. |
| Databáze: | OpenAIRE |
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