Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family

Abstract

The membrane-bound complex of periplasmic permeases comprises two hydrophobic proteins which have been hypothesized to be integral membrane-spaninning proteins. We have investigated the topological organization of the hydrophobic components of the Salmonella typhimurium histidine permease, HisQ and HisM. Both proteins are digested by trypsin and proteinase K when either inside-out or right-side-out membrane vesicles are used. Therefore, these proteins are exposed to both surfaces of the membrane. Digestion with carboxypeptidase and binding studies with antibodies directed against the carboxyl terminus of HisQ and HisM have localized their carboxyl termini to the inside surface of the cytoplasmic membrane. Aminopeptidase digestion suggests periplasmic localization of their amino termini. Alkaline phosphatase fusions to HisQ and HisM indicate the existence of five spanners in both proteins. The periodicity and orientation of spanners and loops in HisQ and HisM match those of the five carboxyl-terminal spanners of MalF, the only other hydrophobic component of the periplasmic permeases for which topological information is available. An alignment of the sequences of all known hydrophobic components of periplasmic permeases is presented which indicates clear conservation of secondary structure and some conservation of primary sequence. The structural conservation of the components is discussed, and a role for a hydrophilic loop containing a conserved sequence (the EAA loop) is proposed.