ABC transporters have long been known to mediate resistance phenotypes in all kingdoms of life, and ATP-driven tripartite efflux pump from Gram-negative bacteria have attracted increasing interest. We give a special focus on MacAB TolC, a prototypical member of the recently described Type VII ABC transporter superfamily, from Escherichia coli. We provide original experimental evidence for the in vitro, substrate-induced ATPase activity and show a maximal activity when the tripartite pump is fully assembled in lipid nanodiscs. These results are evaluated and interpreted in the context of the structural and functional data that have accumulated over the years. •MacAB TolC shows maximal activity when fully assembled and reconstituted in lipids.•MacB is under strict dependance of the hydrophobic environment.•There is an intricate mutual interdependency among MacB, MacA and TolC.•The Periplasmic Core Domain plays a pivotal role in the functioning of the pump.