Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli.

We have examined the importance of the positively charged NH2 terminus of the major outer membrane lipoprotein precursor, prolipoprotein, in the early steps of secretion in Escherichia coli. For this purpose, we have generated three mutants using oligonucleotide-directed mutagenesis in which the charge at the NH2-terminal region was changed from +2 to +1, 0, and -2. The results indicate that the synthesis of prolipoprotein is facilitated by the presence of a positively charged NH2 terminus. In addition, the translocation of prolipoprotein across the cytoplasmic membrane does not absolutely require any basic amino acids at its NH2 terminus. However, the presence of a net negatively charged NH2 terminus causes an initial cytoplasmic accumulation of prolipoprotein which is slowly, post-translationally translocated across the cytoplasmic membrane at a rate which is dependent on the number of positive charges present in this region. The analysis of these mutants clearly demonstrates the importance of the NH2 terminus of the lipoprotein signal peptide in initiating the secretion of this protein in E. coli.