FIGURE 5.

Retro-translocation of CD4 with oxidized disulfide bonds. A, alkylation of CD4-free cysteines. Cells co-transfected with BirA, BAP-CD4, and Vpu were incubated for 5 min at 4 °C in PBS in the presence or absence of 20 mm NEM and rinsed in SDS-lysis buffer. Samples were then run in reducing SDS-PAGE, blotted, and developed with anti-SV5. B, nonreducing and reducing WB-ra of BAP-tagged CD4. Lysates from cells co-transfected with Vpu and treated with MG132 (5 μm for 12 h) were run in nonreducing (left) and reducing (right) conditions and developed with anti-SV5. Biotinylated and nonbiotinylated fractions of glycosylated and deglycosylated CD4 and their oxidation status are indicated with corresponding open and filled arrowheads and arrows. C, WB of biotinylated BAP-tagged CD4. CD4 was immunoprecipitated (IP) with anti-SV5 from cells expressing Vpu in the presence of MG132 (5 μm for 12 h), analyzed in nonreducing (left) and reducing (right) conditions, and developed with HRP-conjugated StrAv. CD4 glycosylation and oxidation status are indicated with open and filled arrows. D, ER-like glycosylation of biotinylated BAP-tagged CD4. WB shows reducing conditions of CD4 immunoprecipitated with anti-SV5 from cells expressing Vpu in the presence or absence of MG132 (5 μm for 12 h) and treated with or without Endo-H. Blot was developed with HRP-conjugated StrAv. E, [³⁵S]methionine labeling of biotinylated CD4. Biotinylated CD4 fraction was immunoprecipitated from cells expressing Vpu after 2-h labeling with [³⁵S]methionine in the presence of MG132 (10 μm), first with anti-SV5 and then with StrAv-conjugated beads, and analyzed in nonreducing (left) and reducing (right) conditions and developed by autoradiography. The glycosylation and oxidation status of biotinylated CD4 bands is indicated.