A proteomic perspective of the mechanism of ABC transporters
ABC transporters are large family of ATP-dependent integral membrane complexes that hydrolyze ATP to translocate substrates against concentration gradient. They are composed of two transmembrane domains (TMDs) that form the translocation pathway and two cytosolic nucleotide-binding domains (NBDs), and a substrate binding protein (SBP), which binds and delivers the substrate to its transporter. While much is known about the molecular-level of mechanism of ABC transporters, little is known of in vivo functional parameters such as their copy number, stoichiometry of the components, localization, and co-localization. In this study we used label-free LC-MS2 to determine these parameters for the 48 E. coli ABC import systems.
Strikingly, we found that the abundances of the SBPs vary, from nM to mM concentrations. We found no correlation between abundances of SBPs and their substrate binding affinities. Rather, we found that the abundances of the SBPs are highly correlated with their affinities towards their cognate transporters.
Further, several observations contrasted with accepted views: For example, some SBPs are absent from the periplasm and present only in the membrane fraction. Their stable membrane association completely depends on their specific interactions with their cognate transporters. Counter-intuitively, in some systems, the copy number of the transporter is 10-100-fold higher than that of its cognate SBP, contrasting with the stoichiometric ratios commonly used in in vitro studies.
Taken together, these in vivo observations suggest that contemporary mechanistic models that were deduced primarily from in vitro data require re-consideration.