A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion

S. Hoffenberg, X. Liu, L. Nikolova, H. S. Hall, W. Dai, R. E. Baughn, B. F. Dickey, M. A. Barbieri, A. Aballay, P. D. Stahl, B. J. Knoll

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)24661-24669
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number32
DOIs
StatePublished - Aug 11 2000

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hoffenberg, S., Liu, X., Nikolova, L., Hall, H. S., Dai, W., Baughn, R. E., Dickey, B. F., Barbieri, M. A., Aballay, A., Stahl, P. D., & Knoll, B. J. (2000). A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion. Journal of Biological Chemistry, 275(32), 24661-24669. https://doi.org/10.1074/jbc.M909600199