Interaction between HIV-1NEF and G(o) proteins in transfected COS-7 cells

Nef protein of HIV/SIV lentiviruses affects G-protein-mediated signaling, a nd physically associates to Lck, a myristoylated and palmitoylated Src-like tyrosine kinase. To assess whether Nef interacts with cu-subunits of heter otrimeric G proteins (G alpha), carrying the same lipidation motif as Lck, we transiently expressed Nef and G(o)alpha (wild-type or nonpalmitoylated C 3S mutant), individually or in combination, in transfected COS-7 cells. Rec ombinant Nef was mostly recovered in particulate fractions, and a Nef-Green Fluorescent Protein chimera was localized at the plasmalemma by in vivo fl uorescence imaging. Moreover, Nef and C3S were entirely solubilized by cold Triton X-100, and excluded from low buoyant density sucrose gradient fract ions, containing caveolin-1, whereas wildtype G(o)alpha was partially resis tant to Triton extraction, and colocalized with caveolin-1. After coexpress ion, Nef recruited soluble C3S to membranes, and the two proteins were coim munoprecipitated by G(o)alpha and Nef antisera. We conclude that Nef intera cts with nonpalmitoylated G(o)alpha, presumably outside caveolin-rich micro domains of the plasma membrane. (C) 2000 Academic Press.