The signal sequence of the murine serine protease inhibitor PAI‐2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild‐type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI‐2 signal sequence. Using a malE signal sequence mutant, which has a Mal‐phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele‐specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG.

The cytosolic and secreted, N‐glycosylated, forms of plasminogen activator inhibitor‐2 (PAI‐2) are generated by facultative translocation. To study the molecular events that result in the bi‐topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI‐2 (mPAI‐2). Interestingly, the six signal sequences we compared (mPAI‐2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI‐2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP‐bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP‐mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.

SecG, an integral membrane component of the Escherichia coli preprotein translocase, contributes to the efficiency of the export process by undergoing cycles of topology inversion in the membrane, coupled with the insertion-deinsertion cycles of SecA. We have previously identified sec alleles of secG that cause a generalized secretion defect. In this study, by screening mutagenized secG libraries for suppressors of a malE signal sequence mutation, we isolated prl alleles of secG. By analogy with secY/prlA, secA/prlD, and secE/prlG, secG could therefore be called secG/prlH. The prlH mutations affect 13 codons distributed along the secG sequence, and none map to the codons affected by sec mutations. prlH suppressors suppress a variety of signal sequence mutations and they allow export of alkaline phosphatase lacking its entire signal sequence. Although secG was not identified in previous selections for prl mutants, several prlH alleles are as strong as the strongest known prlG alleles of secE. Some prlH alleles can also promote the export of alkaline phosphatase fused to predicted cytoplasmic domains of UhpT, an integral membrane protein. These results support the notion that SecG contributes to signal sequence recognition, and suggest that it may also contribute to the topology of integral membrane proteins.

Induction of genes expressed from the arabinose PBADpromoter is very rapid and maximal at low arabinose concentrations. We describe here two mutations that interfere with the expression of genes cloned under arabinose control. Both mutations map to theydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype. One mutation is a large deletion that creates a more efficient −35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT). TheydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose. Disruption of ydeA has no detectable effect on cell growth. Thus, ydeA appears to be nonessential under usual laboratory growth conditions. The ydeA gene encodes a membrane protein with 12 putative transmembrane segments. YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein. Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export. Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect ofydeA transcriptional activation. Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.

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