Identification and characterization of the membrane components of the LIV-I transport system of Escherichia coli.

Abstract

The work described in this thesis concerns the identification, characterization, and expression of the membrane components (LivH, LivM, LivG, and LivF) of the high affinity, periplasmic binding protein-dependent, branched-chain amino acid LIV-I transport system of Escherichia coli. The livH gene has previously been established to be essential for LIV-I transport function. Recombinant DNA methods have permitted the generation of antibody specific to LivH protein, which has assisted in identifying the polypeptide product of the livH gene. Amplified expression of plasmid encoded LivH protein seems to require additional membrane components, LivM and LivG, suggesting they form a complex in the membrane. The identity of the LivH protein is firmly established by amino-terminal and internal amino acid sequence analysis. An amino-terminal block of LivH, believed to be a formyl moiety, must be removed prior to Edman degradation in order to obtain amino-terminal sequence information. A plasmid system was developed that permitted expression and identification of the livM, livG, and livF gene products. The livM gene was demonstrated to be essential to the function of the LIV-I transport system. The LivM protein has been tentatively identified to be a polypeptide band with an apparent Mr of 32 kD when assayed by SDS-PAGE. The identity of the livG gene product was confirmed by amino acid composition and amino-terminal amino acid sequence analysis. A new gene product essential to the function of LIV-I transport system has been designated LivF and was discovered by protein expression studies and subsequently confirmed by nucleotide sequence analysis of the livF gene. The identity of the LivF protein is confirmed by amino acid composition and amino-terminal amino acid sequence analysis. Subcellular fractionation studies establish LivH, LivG and LivF to be integral inner membrane proteins. Regulated expression of the LIV-I proteins was analyzed in vivo by a translational gene fusion between livH and lacZ genes and by expressing plasmid encoded LIV-I genes in minicells. The plasmid encoded expression of the LivH$\sp\prime$-$\sp\prime$LacZ fusion protein and the gene products of the livKHMGF operon is regulated by sub-millimolar concentration of leucine present in the medium.