Role of HRB in Clathrin-dependent Endocytosis

• Published in: The Journal of biological chemistry Vol. 283; no. 49; pp. 34365 - 34373
• Main Authors: Chaineau, Mathilde, Danglot, Lydia, Proux-Gillardeaux, Véronique, Galli, Thierry
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.12.2008; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Line; Cellular Biology; Clathrin - chemistry; Clathrin - physiology; Cloning, Molecular; Dogs; Endocytosis; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Life Sciences; Membrane Transport, Structure, Function, and Biogenesis; Microscopy, Fluorescence; Models, Biological; Neurobiology; Neurons and Cognition; Nuclear Pore Complex Proteins - chemistry; Nuclear Pore Complex Proteins - physiology; Protein Binding; Protein Structure, Tertiary; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - physiology; Subcellular Processes; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M804587200

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.