Fig. 5.

Bafilomycin treatment prevents the turnover of K4Q and S15Y αβ phosphotransferase (A) Transfected HEK293 cells were labeled with TRAN ³⁵S-LABEL methionine/cysteine for 20 min, and αβ phosphotransferase was immunoprecipitated with anti-V5 after a 1- or 8-h chase with or without 0.1 μM bafilomycin a1. The samples were not treated with PNGase F. Bafilomycin treatment increases the level of the β subunits of both WT and K4Q phosphotransferase. (B) Western blot (anti-V5) of HEK293 cells expressing WT, K4Q, or S15Y αβ phosphotransferase treated with or without 0.1 μM bafilomycin a1 for 7 h (representative blot of 2 independent experiments). The mature β subunits of the mutants increase to the level of the WT β subunit after bafilomycin treatment. GAPDH shows equal protein loading. (C) Confocal immunofluorescence microscopy of HeLa cells treated for 4 h with 0.1 μM bafilomycin. The mutants (anti-α subunit) colocalize with GM130 in the cis-Golgi. (Scale bars, 20 μm.)