The amino-terminal charge and core region hydrophobicity interdependently contribute to the function of signal sequences.

We have constructed a series of signal sequence mutants that contain negatively charged amino termini and simplified core regions of varying hydrophobicity levels. This series provides a means of exploring the relative roles of the amino terminus and the hydrophobic core region during transport. The signal peptides with highly hydrophobic core regions support a rapid rate of transport in the presence of a negatively charged amino terminus. We have found that these negatively charged mutants are secreted in a manner similar to the wild-type signal sequence; sodium azide and carbonyl cyanide 3-chlorophenylhydrazone treatments indicate that the negatively charged mutants depend on SecA and the protonmotive force, respectively. These same mutants also demonstrate reduced competition with coexpressed beta-lactamase, reflecting the lower overall affinity for the transport pathway due to the net negative charge at the amino terminus. In addition, the pronounced effects of introducing three negative charges support the conclusion that the two regions function in a concerted manner.