FIGURE 5.

Analysis of biogenesis kinetics of the cytoplasmic membrane proteomes of Ffh-depleted and control cells by two-dimensional BN/SDS-PAGE and the protein synthesis in whole cells. A, WAM121 cells grown in the presence (control) and absence (depleted of Ffh for 4 h) of arabinose were labeled with [³⁵S]methionine for 30 s (top) and labeled for 30 s followed by a chase for 5 min with cold methionine (bottom). The cytoplasmic membrane fractions were isolated by density centrifugation from a mixture of labeled and nonlabeled cells as described under “Experimental Procedures.” The cytoplasmic membrane fractions were analyzed by two-dimensional BN/SDS-PAGE and phosphorimaging. Representative two-dimensional BN/SDS-polyacrylamide gels with proteins detected by phosphorimaging (protein incorporation) are shown. Notably, the 30-s pulse and the 5-min chase experiments represent two different experiments. B, WAM121 cells grown in the presence (control) and absence (depleted of Ffh for 4 h) of arabinose were labeled with [³⁵S]methionine for 30 s. Total cell lysates (0.02 A₆₀₀ unit) were analyzed by one-dimensional SDS-PAGE and phosphorimaging. The intensities of with [³⁵S]methionine-labeled proteins were quantified using Image Gauge 3.4 software (Fuji).