Amino acid residues in the pro region of Escherichia coli heat-stable enterotoxin I that affect efficiency of translocation across the inner membrane

Escherichia coli heat-stable enterotoxin 1p (ST1p), which is a typical extracellular toxin consisting of 18 amino acid residues, is synthesized as a precursor consisting of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. Though the pre region functions as a conventional leader peptide that guides the following region to cross the inner membrane, the role of the pro region in the maturation pathway remains to be elucidated. We previously indicated that the sequence from residues 29 to 38 in the pro region increases the efficiency of STI translocation across the inner membrane (H. Yamanaka, Y. Fuke, S. Hitotsubashi, Y. Fujii, and K. Okamoto, Microbiol. Immunol. 37:195- 205, 1993). We therefore examined the amino acid residues in the sequence that are responsible for this function. We substituted several amino acid residues in the sequence by means of oligonucleotide-directed site-specific mutagenesis. We then evaluated the effect of the substitution on the efficiency of STI translocation across the inner membrane by determining the enterotoxic activity of the culture supernatant, the amount of a fusion protein consisting of STI and nuclease A released into the periplasm, and the amount of the labeled ST released into the periplasm after pulse-labeling with [³⁵S]cysteine. Substitution of the charged amino acid residues at positions 29 to 31 (K-E-K) with hydrophobic (I-V-L, F-W-F, or F-W-Q) or basic (K-K-K) residues significantly reduced these values in every assay. In contrast, the substitution of these amino acid residues with acidic amino acid residues (E-E-E) increased these values in all assays. This means that the negative charge near position 30 is important for STI to translocate efficiently across the inner membrane. A similar substitution of lysine residues at positions 37 and 38 showed that they are not involved in the translocation of STI across the inner membrane.