Interaction between HIV-1 NEF and G(o) proteins in transfected COS-7 cells.
Nef protein of HIV/SIV lentiviruses affects G-protein-mediated signaling, and physically associates to Lck, a myristoylated and palmitoylated Src-like tyrosine kinase. To assess whether Nef interacts with alpha-subunits of heterotrimeric G proteins (Galpha), carrying the same lipidation motif as Lck, we transiently expressed Nef and G(o)alpha (wild-type or nonpalmitoylated C3S mutant), individually or in combination, in transfected COS-7 cells. Recombinant Nef was mostly recovered in particulate fractions, and a Nef-Green Fluorescent Protein chimera was localized at the plasmalemma by in vivo fluorescence imaging. Moreover, Nef and C3S were entirely solubilized by cold Triton X-100, and excluded from low buoyant density sucrose gradient fractions, containing caveolin-1, whereas wild-type G(o)alpha was partially resistant to Triton extraction, and colocalized with caveolin-1. After coexpression, Nef recruited soluble C3S to membranes, and the two proteins were coimmunoprecipitated by G(o)alpha and Nef antisera. We conclude that Nef interacts with nonpalmitoylated G(o)alpha, presumably outside caveolin-rich microdomains of the plasma membrane.
Guzzi
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F
Department of Experimental and Environmental Medicine and Biotechnology, School of Medicine, University of Milano-Bicocca, Via Cadore 48, Monza, 20052, Italy.
Celano
E
E
Levi
G
G
Parenti
M
M
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D015693:Gene Products, nef / Q000276:immunology / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000378:metabolism*; D007106:Immune Sera; D008164:Luminescent Proteins / Q000378:metabolism; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D015693:Gene Products, nef; D007106:Immune Sera; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins