IN-VITRO DISASSEMBLY AND REASSEMBLY OF AN ABC TRANSPORTER, THE HISTIDINE PERMEASE

The membrane-bound complex of the Salmonella typhimurium periplasmic h istidine permease, a member of the ABC transporters (or traffic ATPase s) superfamily; is composed of two Integral membrane proteins, HisQ an d HisM, and two copies of an ATP-binding subunit, HisP. The complex hy drolyzes ATP upon induction of the activity by the liganded soluble re ceptor, the periplasmic histidine-binding protein, HisJ, Here we take advantage of the modular organization of this system to show that the nucleotide-binding component can be stripped off the integral membrane components, HisQ and HisM, The complex can be reconstituted by using the HisP-depleted membranes containing HisQ and HisM and pure soluble HisP, We show that HisP has high affinity for the HisP-depleted comple x, HisQM, and that two HisP molecules are recruited independently of e ach other for each HisQM unit, The in vitro reassembled complex has en tirely normal properties, responding to HisJ and ATPase inhibitors wit h the same characteristics as the original complex and in contrast to those of soluble HisP, These results show that HisP is absolutely requ ired for ATP hydrolysis, that HisQM cannot hydrolyze ATP, that HisP de pends on HisQM to relay the inducing signal from the soluble receptor, HisJ, and that HisQM regulates the ATPase activity of HisP, We also s how that HisP changes conformation upon exposure to phospholipids.