SUPPRESSION OF SIGNAL SEQUENCE DEFECTS AND AZIDE RESISTANCE IN ESCHERICHIA-COLI COMMONLY RESULT FROM THE SAME MUTATIONS IN SECA

The SecA protein of Escherichia call is required for protein translocation from the cytoplasm. The complexity of SecA function is reflected by missense mutations in the secA gene that confer several different phenotypes: (i) conditional-lethal alleles cause a generalized block in protein secretion, resulting in the cytoplasmic accumulation of the precursor forms of secreted proteins; (ii) azi alleles confer resistance to azide at concentrations up to 4 mM; and (iii) pr1D alleles suppress a number of signal sequence mutations in several different genes. To gain further insights into the role of SecA in protein secretion, we have isolated and characterized a large number of pr1D mutations, reasoning that these mutations alter a normal function of mild-type SecA. Our results reveal a striking coincidence of signal sequence suppression and azide resistance: the majority of pr1D alleles also confer azide resistance, and all azi alleles tested are suppressors. We suggest that this correlation reflects the mechanism(s) of signal sequence suppression. There are two particularly interesting subclasses of pr1D and azi alleles. First, four of the pr1D and azi alleles exhibit special properties: (i) as suppressors they are potent enough to allow Pr1D (SecA) inactivation by a toxic LacZ fusion protein marked with a signal sequence mutation (suppressor-directed inactivation), (ii) they confer azide resistance, and (iii) they cause modest defects in the secretion of wild-type proteins. Sequence analysis reveals that all four of these alleles alter Tyr-134 in SecA, changing it to Ser, Cys, or Asn. The second subclass consists of seven pr1D alleles that confer azide supersensitivity, and sequence analysis reveals that six of these alleles are changes of Ala-507 to Val. Both of the affected amino acids are located within different putative ATP-binding regions of SecA and thus may affect ATPase activities of SecA. We suggest that the four azide-resistant mutations slow an ATPase activity of SecA, thus allowing successful translocation of increased amounts of mutant precursor proteins.