EFFECT OF LYS(-20) SUBSTITUTIONS IN ALKALINE-PHOSPHATASE SIGNAL PEPTIDE ON SECRETION OF THE ENZYME

The effect of substitutions for positively charged Lys(-20) in the N-t erminal domain of the E. coli alkaline phosphatase signal peptide on t he enzyme secretion has been studied. The amber-suppressor method was used to obtain mutant alkaline phosphatases: amber mutation was introd uced in the appropriate position of the alkaline phosphatase gene usin g oligonucleotide-directed mutagenesis, and mutant proteins were synth esized in E. coli strains producing amber-suppressor tRNAs specific fo r Tyr, Gly, Ala, Glu, Phe, His, Cys, and Pro, All the mutant proteins can be translocated across the cytoplasmic membrane and assembled in t he periplasm to active enzyme molecules. However, certain amino acid s ubstitutions reduce the rate of protein maturation. Their effect depen ds not only on the charge of the amino acid residue, but also on its n ature. Thus, introduction of positively charged His and the polar unch arged Tyr has no significant effect, while introduction of negatively charged Glu and the hydrophobic Ala, Phe, and Pro, as well as Gly and Cys, has an inhibiting action. The data show the important role of the primary structure of the signal peptide N-terminus in secretion.