FIGURE 4.

Cys-245 is involved in dimerization of the γ-subunits. A, cell extracts from COS-7 cells overexpressing the human wild-type (WT) and mutant (C142S, C157S, C169S, and C245S) γ-subunits were incubated for 1 h in the presence (+) or absence (−) of PNGase F. The proteins were separated by SDS-PAGE under reducing (+ ME) and nonreducing (− ME) conditions and analyzed by γ-subunit Western blotting. The asterisk indicates an unspecific band. B, the mutant C245S γ-subunit C-terminally fused to GFP shows colocalization with the cis-Golgi apparatus marker protein GM130 in BHK cells. Scale bar = 10 μm. Magnified views of the indicated white squares are shown below. C, cell extracts (75 μg) and media conditioned for 24 h (15%) from COS-7 cells overexpressing the human wild-type (WT) and mutant C245S (C) γ-subunits were analyzed by γ-subunit Western blotting.