Figure 3.

All of the polyleucine H-segments are recognized and processed by SecA, but full incorporation into the inner membrane (IM) requires 14 leucines. This suggests a translocon/membrane partitioning process occurs as the secreted protein passes through the translocon. As in Figure 2, this immunoblot identifies T7 tags, which are contained within the N-terminal domain carrying the H-segment. The appearance of the lower molecular weight bands indicates cleavage by SPase I and therefore processing of the construct by SecA. In lanes 10L to 12L, the fragments are found only in the soluble (C+P=cytoplasm plus periplasm) fraction. Beginning with 14L, all fragments are found in the IM fraction. Note the fragment molecular weight differences for lanes 14L-16L compared with lanes 10L-12L. The difference is particularly obvious in 13L, which reveals clearly the transition from the cytoplasm to inner membrane. The difference in molecular weight arises from processing by the RseP intramembrane protease (see Figure 4). The C-H-F constructs were expressed in E. coli BL21 cells grown at 37 °C in SOC media augmented with glucose, MgCl₂, and MgSO₄. See Materials and Methods.