A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion.
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.
Hoffenberg
S
S
Departments of Medicine (Pulmonary Division), Molecular Physiology and Biophysics, Microbiology and Immunology, and Molecular and Cellular Biology, Baylor College of Medicine and Houston Veterans Affairs Medical Center, Houston, Texas 77030, USA.
Liu
X
X
Nikolova
L
L
Hall
H S
HS
Dai
W
W
Baughn
R E
RE
Dickey
B F
BF
Barbieri
M A
MA
Aballay
A
A
Stahl
P D
PD
Knoll
B J
BJ
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D011992:Endosomes / Q000502:physiology* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology / Q000648:ultrastructure; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013873:Thionucleotides / Q000378:metabolism; D014162:Transfection; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C413025:SUN2 protein, human; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D020696:rab5 GTP-Binding Proteins