Characterization of the internal signal-anchor domain of Escherichia coli leader peptidase.

Leader peptidase, an integral transmembrane protein of Escherichia coli, is synthesized without a cleavable amino-terminal leader peptide. Of the five domains that participate in the membrane assembly of this protein, one is an internal "signal" region. We have used oligonucleotide-directed mutagenesis to examine the properties of the internal signal that are crucial for leader peptidase assembly. For this purpose, the net charge at the amino terminus of the internal signal was changed from +2 to +1 and -1 and, at the carboxyl terminus of the signal, from 0 to -1 or +1. These mutations had no effect on the membrane assembly of leader peptidase, suggesting that the charges have little role in the signal function. The apolar core of this signal was disrupted by substitution of basic amino acids for apolar residues. Substitution of an arginyl residue at position 70, or two arginyl residues at position 67 and 69, prevented membrane assembly. However, substitution of an arginyl residue at position 66 or either arginyl or lysyl residue at position 68 was without effect. Thus, while the apolar character of the internal signal is important, the precise position of a charged residue determines its effect on assembly.