The histidine periplasmic permease of Salmonela typhimuium has been partially purified and reconstituted into proteoliposomes. In this in vitro preparation, transport activity is completely dependent on the presence of all four permease proteins (HisJ, HisQ, HisM, and HisP) and on internal ATP. The reconstituted system shows initial rates of transport that are comparable to those obtained with right-side-out membrane vesicles and it establishes a 100-fold concentration gradient for histidine. have been hypothesized to be involved in the coupling ofATP to substrate transport (6, 7). Interestingly, proteins homologous to this family have been discovered also in eukaryotic organisms where they are also involved in transport-related processes, such as in the multidrug-resistance excretion mechanism (MDR) and in the transport and deposit of the Drosophila eye pigment (8,9).Our studies utilize as a model system the well-characterized histidine permease of Salmonella typhimurium (10), which is composed of the histidine-binding protein, HisJ, two hydrophobic integral membrane proteins, HisQ and HisM, and the nucleotide-binding membrane protein, HisP. Data obtained using intact cells implicated ATP as the energy source, and it was shown incontrovertibly that the electrochemical gradient is not involved in energization (3). In vitro studies were also initiated in order to approach the question of the proximal energy source from a biochemical point of view and to advance our understanding of the molecular mechanism of action of these permeases. With an in vitro system that utilizes right-side-out membrane vesicles to which the histidine-binding protein is added externally (11,12), additional evidence was obtained that energization is derived from the ATP generated from the electrochemical gradient via the proton-conducting FoF1 ATPase; it was also shown that the liganded binding protein is the true substrate in transport, interacting with the membrane complex with a Km of 65 j.M (12). A second in vitro system, which consists of inside-out membrane vesicles containing entrapped histidine-binding protein and energized by externally added ATP, but not by a nonhydrolyzable analog of ATP, also supplied evidence that ATP is the proximal energy source and that its hydrolysis is necessary (13). However, both above systems are unsatisfactory for further biochemical studies, since the only pure protein utilized is HisJ, the others being still embedded in membrane vesicles. We now describe a third system, reconstituted proteoliposomes.Protedliposome reconstitution of periplasmic permeases has not been previously attempted, presumably because of the complex multicomponent nature of these systems and of the uncertainty as to their mechanism of energization. In addition, the methodology for reconstituting bacterial permeases in general was not available until fairly recently (14-17). Armed with the availability of the overproduction of our permease membrane proteins, with the presumption that ATP is the energy source, and with t...

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The region of the colicin El polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin El against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical

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