Figure 4.

N-terminal-extended native secreted proteins (MalE, OmpA, and PhoA) can serve as SecA targets. SecA targeted all constructs to the membrane, based upon SPase I cleavage. The cleaved T7-labeled fragments revealed two bands on the immunoblots resulting from cleavage by SPase I (left-hand lanes labeled RseP⁺). These bands arise from post-cleavage processing by the site-2 intramembrane metalloprotease RseP system [19, 20], as proven by the presence of a single-band in RseP⁻ mutants (right-hand, single bands). These results indicate that less stable TM segments are attacked by RseP. However, RseP processes membrane embedded sequences only if they have been cleaved initially by SPase I. See Materials and Methods for descriptions of the RseP⁻ cells.