Figure 5

Vpu-mediated CD4 degradation involves dislocation of ubiquitinated CD4 conjugates from the ER membrane to the cytosol. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10μg of envelope-defective provirus (HxBc2-pr^-, vpu^-, env^- or HxBH10-pr^-, vpu⁺, env^-) and 15 μg of his(6)/c-myc-Ub K48/R expression plasmid where indicated. Cells were treated with BFA for 2 h before mechanical lysis. CD4-Ub conjugates were immunoprecipitated with anti-myc monoclonal antibodies prior to western-blot analysis with anti-CD4 polyclonal antibodies while control proteins in each fraction were revealed by western-blot. Actin and calnexin were used as cytosolic and membrane controls, respectively. A. Membrane (M) and cytosolic (C) fractions were separated and treated as described in the materials and methods section. B. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area of the autoradiogram that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Relative levels of ubiquitinated CD4 conjugates were determined as described in the legend of Fig. 2B. Error bars reflect standard deviations from duplicate independent experiments. C. Membrane (M) fractions were treated with Na₂CO₃ (pH 11) as described in materials and methods. Treated membrane and supernatant (S) were subsequently recovered by centrifugation. Fractions were analyzed as described above in A. D. Quantitative analysis of the relative amounts of ubiquitinated CD4 molecules (as described in the legend of Fig. 2B) present in each fraction relative to the amounts measured in absence of Vpu (arbitrarily set at 1). (asterisk) represents the area that was used for the quantitation of CD4-Ub conjugates. Non-specific background signal detected in lanes 7 and 8 was subtracted. Error bars reflect standard deviations from duplicate independent experiments.