The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOXl plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine-isoleucine-valine binding protein and the leucine-specific binding protein are synthesized as precursors with 23 amino acid N-terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine-isoleucine-valine binding protein, it and the closely related leucine-specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N-terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans-positive side of the membrane, thus providing an additional role for the transmembrane potential.

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.