Strains of Escherichia coli in which lacZ (specifies beta-galactosidase) is fused to genes that specify exported proteins such as LamB (lambda receptor) exhibit unusual phenotypes. In particular, such strains are killed by high-level expression of the LacZ hybrid protein. Previous results suggest that this overproduction phenotype is the consequence of a lethal jamming of the cellular protein export machinery and this hypothesis is supported by the observed accumulation of the precursor forms of many noncytoplasmic proteins within the moribund cell. Under conditions in which protein export is compromised, biochemical and immunocytochemical analyses indicate that these hybrid proteins can be found in transmembrane orientation. To identify the cellular component rendered rate-limiting by the LacZ hybrid protein under jamming conditions we have utilized signal sequence mutations, which block entry of the hybrid protein into the export pathway, and a dominant suppressor of these lesions, prlA4. Data obtained with a series of merodiploids heterozygous and homozygous for prlA+ and prlA4 show that PrlA is the component sequestered by hybrid jamming. Taken together, these results suggest that PrlA is a component of the export machinery that functions in the translocation of proteins across the cytoplasmic membrane.
The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.
The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.
By fusing the transcriptional and translational start signals of lacZ to envZ, we have obtained high-level synthesis of a truncated EnvZ protein (EnvZ115) in which the first 38 amino acids of EnvZ are replaced with the first 8 amino acids of LacZ. Using this construct, we have partially purified the EnvZ115 protein and demonstrated that this protein can be phosphorylated in vitro. We suggest that phosphorylation may be an important feature of EnvZ function.
During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane. Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).
We exploited the conditional-lethal phenotype of secB null mutations to demonstrate that SecB function was required for PrlA-mediated suppression of signal sequence mutations. The results of these experiments provide information about the functions performed and the sequence determinants recognized by each of these components of the protein export machinery of Escherichia coli.
Our laboratory has been utilizing the Escherichia coli outer membrane protein LamB to study the mechanism of protein localization. Various lines of evidence suggest that, in addition to a signal sequence, regions within the mature protein are required for efficient localization. In particular, studies using LamB-LacZ hybrid proteins have identified regions between amino acids 27 and 49 of mature LamB, which may play an important role in localization. To elucidate further the function of these regions, a series of in-frame deletions that remove varying lengths of early lamB sequences was constructed. The effects of these deletions on export of a large LamB-LacZ hybrid protein, 42-1, and on export of an otherwise wild-type LamB protein were determined. We find a strong correlation between the sequences deleted and the export phenotypes these deletions impart to both LamB and the LamB-LacZ42-1 hybrid protein. On the basis of these findings, the deletions can be divided into several distinct classes that define a region within mature LamB that participates in localization. This region extends amino terminally from amino acid 28 of the mature protein and functions in the rapid and efficient localization of LamB from the cytoplasm.