The PlcH R9K and PlcH R10K signal sequence mutants mislocalize to the inner membrane. (A) PlcH activity in supernatants (S) and bacterial fractions. Error bars indicate standard errors of the means of three independent experiments. (B and C) Total PlcH protein in supernatants (B) and inner membrane fractions (C). Lane 1, purified PlcH enzyme, used as size marker for mature PlcH (the lower band represents nonspecific degradation product); lane 2, ADD1976 with the vector; lane 3, ADD1976 with wild-type PlcH; lane 4, ADD1976 with PlcH R9K mutant; lane 5, ADD1976 with PlcH R10K mutant; lane 6, ADD1976 with PlcH R9K spheroplast. The arrows and arrowhead in lanes 1 (B and C) and 4 (C) show the different forms of PlcH.