Abstract
We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the “nonessential activator model” where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function. The increased ATPase rates in the presence of GSSG more than compensate for the modest negative cooperativity observed between MgATP and GSSG in lipid environments. These studies highlight the central role of the elusive ternary complex in accelerating the ATPase rate that is at the heart of coupling mechanism between substrate transport and ATP hydrolysis.
Highlights
The ability of substrates to stimulate the ATPase activity of ATP binding cassette (ABC) transporters has been utilized to identify potential substrates transported by members of this ubiquitous family.[1,2,3,4] Despite the practical significance of this approach for identifying the substrates of ABC exporters, more detailed kinetic characterizations of the ATPase activity as a function of the concentrations of both MgATP and transported substrate have not, to our knowledge, been reported
We have previously established that oxidized glutathione (GSSG) can be transported by NaAtm1.13 Together with structural analyses that have captured multiple conformational states, a framework for an alternating access transport cycle has been defined, with the interconversion between states coupled to the binding and hydrolysis of ATP.[13,14]
As a first step to resolving this paradox, we characterized the dependence of the steady state ATPase kinetics on the concentrations of MgATP and the transported substrate, oxidized glutathione (GSSG), for the ABC exporter NaAtm[1]
Summary
The ability of substrates to stimulate the ATPase activity of ATP binding cassette (ABC) transporters has been utilized to identify potential substrates transported by members of this ubiquitous family.[1,2,3,4] Despite the practical significance of this approach for identifying the substrates of ABC exporters, more detailed kinetic characterizations of the ATPase activity as a function of the concentrations of both MgATP and transported substrate have not, to our knowledge, been reported. As a first step to resolving this paradox, we characterized the dependence of the steady state ATPase kinetics on the concentrations of MgATP and the transported substrate, oxidized glutathione (GSSG), for the ABC exporter NaAtm[1].
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