Expression and purification of COMATOSE, a plant peroxisomal ABCD transporter, for functional and structural studies

ABC transporters represent one of the largest and most diverse protein superfamilies and are found in all known organisms. They couple the hydrolysis of ATP to the transport of different substrates across various biological membranes reflecting their critical role in many different cellular functions. The peroxisomal family D ABC transporter COMATOSE (CTS), which is expressed in Arabidopsis thaliana, is responsible for transporting acyl-CoAs into the peroxisome where they are used in beta oxidation. Importantly, the transporter possesses intrinsic thioesterase activity whereby the acyl-CoA substrates are cleaved during transport across the peroxisomal membrane. While this activity has been demonstrated biochemically, its nature remains unclear due to a lack of structural data on CTS. Reported here is the development and optimisation of purification protocols suitable for structure determination of CTS using cryo-EM, as well as the successful reconstitution of the purified and active transporter into proteoliposomes. Optimisation of the solubilisation and purification of an ATP-hydrolysis deficient mutant (CTSD606N/E607Q) enabled the first 3D reconstruction of CTSD606N/E607Q, revealing the transporters overall fold from a low-resolution map, and confirmed the steps required for generating a high-resolution structure in future studies. Using the same purification method, the wild-type and mutant were reconstituted into proteoliposomes where CTS showed substrate stimulated ATPase activity, opening the possibility of studying the transporter in a totally isolated system. These results pave the way for future studies on CTS to place the biochemistry of thioesterase activity into a structural framework and understand the transport cycle as well as the transporters broad substrate specificity. This work will synergise with the growing body of work on other family D members, such as human ABCD1 and yeast Pxa1p/Pxa2p, and help to explain early CTS observations
in its native A. thaliana, thereby contributing to a more comprehensive understanding of this intriguing family of ABC transporters.