FIGURE 6.

Cysteine mutants with reduced charge density show altered oligomeric profiles. Lanes 1, WT; lanes 2, C52S; lanes 3, C124S; lanes 4, C260S; lanes 5, C192S; lanes 6, C196S; lanes 7, C192S/C196S; lanes 8, C381S; lanes 9, C395S; lanes 10, C415S; lanes 11, C679S; lanes 12, untransfected HEK. A, serine replacement mutants (Figs. 3–5) were analyzed by Western blot with equal total protein loading verified by reprobing for actin. Mutants showing V_½ shift or z increase showed normal oligomeric populations compared with WT prestin. Unglycosylated monomer, glycosylated monomer, and dimer bands are clearly within the resolving capacity of the gel and are labeled as M, G, and D, respectively. Higher order bands, probably representing the trimer and tetramer, are labeled T and Te, respectively. Charge density mutants, C192S/C196S and C415S, showed qualitatively reduced glycosylated monomer levels. B, the glycosylated monomer bands of serine replacement mutants were quantified from membrane fractions isolated on sucrose gradients. Equal total membrane protein loading was assessed by measuring APP intensity. C, the intensity of the glycosylated monomer band normalized to the intensity of APP (gray bars) and mean charge density (dotted line with S.E. bars and n values) is plotted for each lane of B. Prestin mutants are displayed in order of decreasing charge density, with the corresponding lane numbers of B shown in parentheses. C192S/C196S, C415S, C196S, and C192S all show significantly reduced charge density compared with wild type (p < 0.05). D, total prestin levels were quantified from purified, biotinylated membrane protein fractions. Mutants with levels of glycosylated monomer (gray bars) equal to or greater than that of WT were also found to have WT levels of total membrane-localized prestin (black bars).