GENETIC-ANALYSIS OF AN ESSENTIAL CYTOPLASMIC DOMAIN OF ESCHERICHIA-COLI SECY BASED ON RESISTANCE TO SYD, A SECY-INTERACTING PROTEIN

We previously described a dominant negative secY(-d)1 allele in Escher ichia coli, whose product interferes with protein export, presumably b y sequestering SecE, the stabilizing partner of SecY. Syd is the produ ct of a multicopy suppressor of the secY(-d)1 phenotype, and its overp roduction preferentially stabilizes the wild-type SecY protein. In con trast, overproduction of Syd is toxic to the secY24 mutant, which show s a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240-->Asp). The secY249 mutati on (Ala249-->Val) intragenically suppressed Syd sensitivity, but not t he temperature-sensitive Sec phenotype of the secY24 mutation. The Sec Y249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The oth er two mutations changed residue 240 (the site of the secY24 alteratio n) to Asn (secY245) or Ala (secY241) and restored the ability of the c ell to export protein. Although the secY245 mutant retained some sensi tivity to Syd overproduction, the secY241 mutant was completely Syd-re sistant. Furthermore, the secY241 mutation seemed to represent a ''hyp er reversion'' with respect to the SecY-SecE interaction. Protein expo rt in this mutant was no longer sensitive to SecY(-d)1. When the secY( -d)1 mutation was combined intragenically with sec Y241, the resulting double mutant gene (secY(-d)1-241) showed an increased ability to int erfere with protein export. On the basis of our model for SecY(-d)1, t hese results suggest that the secY241 alteration enhances SecY-SecE in teraction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE req uired for the establishment of the translocase complex.