PROCESSING OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE - ROLE OF THE PRIMARY STRUCTURE OF THE SIGNAL PEPTIDE CLEAVAGE REGION

A wide range (69) of mutant Escherichia coli alkaline phosphatases wit h single amino acid substitutions at positions from -5 to +1 of the si gnal peptide were obtained for studying Protein Processing as a functi on of the primary structure of the cleavage region. Amber suppressor m utagenesis, used to create mutant proteins, included: (i) introduction of amber mutations into respective positions of the phoA gene; and (i i) expression of each mutant phoA allele in E. coli strains producing amber suppressor tRNAs specific to Ala, Cys, Gin, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser and Tyr. Most amino acid substitutions at positions -3 and -1 resulted in a complete block of protein processing. These d ata give new experimental support for the ''-3, -1 rule''. Only Ala, G ly and Ser at Position -1 allowed protein processing, and Ala provided the highest rate of processing. The results revealed the more conserv ative nature of the amino acids at the -1 position of signal peptides of Gram-negative bacteria as compared with those of eukaryotic organis ms. Position -3 was less regular, since not only Ala, Ser and Gly, but also Leu and Cys at this position, allowed the processing. Mutations at position -4 had an insignificant effect on the processing. Surprisi ngly, efficient processing was provided mainly by large amino acid res idues at position -2 and by middle-sized residues at position -5, indi cating that the processing rate is affected by the size of amino acid residues not only at positions -1 and -3. Conformation analysis of the cleavage site taken together with the mutation and statistical data s uggests an extended p-conformation of the -5 to -1 region in the signa l peptidase binding pocket. (C) 1998 Academic Press Limited.