In vitro disassembly and reassembly of an ABC transporter, the histidine permease.
The membrane-bound complex of the Salmonella typhimurium periplasmic histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP. The complex hydrolyzes ATP upon induction of the activity by the liganded soluble receptor, 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 complex, HisQM, and that two HisP molecules are recruited independently of each other for each HisQM unit. The in vitro reassembled complex has entirely normal properties, responding to HisJ and ATPase inhibitors with the same characteristics as the original complex and in contrast to those of soluble HisP. These results show that HisP is absolutely required for ATP hydrolysis, that HisQM cannot hydrolyze ATP, that HisP depends on HisQM to relay the inducing signal from the soluble receptor, HisJ, and that HisQM regulates the ATPase activity of HisP. We also show that HisP changes conformation upon exposure to phospholipids.
Liu
P Q
PQ
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Ames
G F
GF
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D001693:Biological Transport, Active; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D010743:Phospholipids; C043828:histidine permease, Bacteria