We present an overview of the architecture of ATPbinding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors/regulators, and assemble into macromolecular complexes with specific properties.

Cystathionine-␤-synthase (CBS) domains are found in >4,000 proteins in species from all kingdoms of life, yet their functions are largely unknown. Tandem CBS domains are associated with membrane transport proteins, most notably members of the ATPbinding cassette (ABC) superfamily; voltage-gated chloride channels and transporters; cation efflux systems; and various enzymes, transcription factors, and proteins of unknown function. We now show that tandem CBS domains in the osmoregulatory ABC transporter OpuA are sensors for ionic strength that control the transport activity through an electrostatic switching mechanism. The on͞off state of the transporter is determined by the surface charge of the membrane and the internal ionic strength that is sensed by the CBS domains. By modifying the CBS domains, we can control the ionic strength dependence of the transporter: deleting a stretch of C-terminal anionic residues shifts the ionic strength dependence to higher values, whereas deleting the CBS domains makes the system largely independent of ionic strength. We present a model for the gating of membrane transport by ionic strength and propose a new role for CBS domains.ATP-binding cassette transporter ͉ cystathionine-␤-synthase domains ͉ cell volume regulation ͉ ionic strength sensor ͉ osmoregulation C

Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomeric OpuA complexes with only one SBD or one functional and one non-functional SBD (inactivated by covalent linkage of a substrate mimic) have been constructed, and the substrate binding and transport kinetics of the puri®ed transporters, reconstituted in liposomes, have been determined. The data indicate that the two SBDs of OpuA interact in a cooperative manner in the translocation process by stimulating either the docking of the SBDs onto the translocator or the delivery of glycine betaine to the translocator. It appears that one of these initial steps, but not the later steps in translocation or resetting of the system to the initial state, is rate determining for transport. These new insights on the functional role of the extracytoplasmic SBDs are discussed in the light of the current knowledge of substrate-binding-protein-dependent ABC transporters.

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine ␤-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mah

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