Figure 4.

Mechanism of SNAT1 Depletion by Vpu

(A) Interaction of SNAT1 with Vpu. HeLa cells stably transduced with Vpu-HA were immunoprecipitated with anti-SNAT1 (G63; first panel) or anti-HA (second panel) antibodies and immunoblotted with anti-SNAT1 (H60) or anti-Vpu antibodies. Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls.

(B) Ubiquitination of SNAT1 by Vpu. HeLa cells stably transduced with Vpu-HA were either immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (first panel) or immunoprecipitated with anti-SNAT1 (G63) antibody, re-immunoprecipitated with anti-SNAT1 (H60) antibody, and immunoblotted with anti-SNAT1 (H60) and anti-ubiquitin antibodies (second panel). Untransduced HeLas transfected with SNAT1-specific siRNA were included as controls. Ubiquitinated SNAT1 in control (blue arrow) and Vpu-expressing (red arrow) HeLas is highlighted.

(C) β-TrCP-dependent depletion of SNAT1. HeLa cells stably transduced with Vpu-HA were transfected with control or β-TrCP-specific siRNA then immunoblotted.

(D and E) SNAT1 depletion via an endolysosomal pathway. HeLa cells stably transduced with Vpu-HA were either treated with MG132, lactacystin, concanamycin, or bafilomycin (D) or transfected with control or TSG101-specific siRNA (E) then immunoblotted.

(F) Molecular determinants of SNAT1 downregulation. Jurkats stably expressing Vpu WT or indicated Vpu mutants were immunoblotted. Cells transduced with empty vector (blue), Vpu WT (red), and Vpu A14L (pink) are highlighted. The same cells stained with anti-CD4 or anti-tetherin antibodies and analyzed by flow cytometry are shown in Figure S5A.

See also Figure S5.