Role of HRB in clathrin-dependent endocytosis.
Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.
Chaineau
Mathilde
M
"Membrane Traffic in Neuronal and Epithelial Morphogenesis," INSERM Avenir Team and Institut Jacques Monod, CNRS UMR7592, Universities Paris 6 and Paris 7, Paris, F-75005 France.
Danglot
Lydia
L
Proux-Gillardeaux
Véronique
V
Galli
Thierry
T
D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000737:chemistry / Q000502:physiology*; D003001:Cloning, Molecular; D004285:Dogs; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D020798:Two-Hybrid System Techniques
C119724:AGFG1 protein, human; D002966:Clathrin; D028861:Nuclear Pore Complex Proteins; D016601:RNA-Binding Proteins; D049452:Green Fluorescent Proteins