Studies on Escherichia coli membrane protein biogenesis: Mechanism of signal peptide peptidase A and the influence of YidC depletion on cellular processes

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.;YidC depletion affects membrane protein insertion and leads to a defect in the growth of the E. coli cell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions. We used a gene-chip method to compare the transcriptome of JS71 (control) and JS7131 (yidC depletion strain). Of the more than 4300 identified genes, 163 were up-regulated and 99 genes down-regulated upon YidC depletion, including genes which are responsible for DNA/RNA repair, energy metabolism, various transporters, proteases and chaperones, stress response and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found up-regulation of the genes encoding the energy transduction proteins F1Fo ATP synthase and cytochrome bo3 oxidase due to perturbation in the assembly when YidC was depleted. We also determined that the high level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore CCCP which dissipates the proton motive force. In addition, the gene chip data reveals the Cpx stress pathway is activated upon YidC depletion. The data provides better understanding toward YidC's function for a number of celluar processes.