The yeast cadmium factor 1 protein (Ycf1p) is an ATP-binding cassette (ABC) transporter that is located in the vacuolar membrane and is responsible for transporting glutathione-conjugated metals from the cytoplasm into the vacuole. Ycf1p contains the ABC core structure of two transmembrane domains (TMD1, TMD2) and two nucleotide-binding domains (NBD1, NBD2). As a member of the C-subfamily of ABC proteins (ABCC), Ycf1p also contains an N-terminal extension comprised of an additional TMD (TMD0) and L0 linker. Although high-resolution structures of many ABC transporters have been determined, the NBDs can be at low resolution in cryo-EM maps. Thus, studies of the isolated cytosolic NBDs are crucial for obtaining molecular-level details of the dynamics of these catalytic entities, for example. In this study, we present a scheme for obtaining samples of NBD2 from the yeast cadmium factor 1 protein (Ycf1p) in a soluble, monomeric, and functional form. While production of NBD1 from Ycf1p and other ABC proteins has been accomplished, generating samples of NBD2 from different ABC proteins has been elusive for the most part, particularly for ABCC proteins. We show that NBD2 preparation necessitates minimizing dimerization and aggregation of the protein at multiple steps during the purification, which is accomplished by employing a solubility tag, eliminating nucleotides from the buffers, and limiting the duration of spin concentrating steps. This work lays the foundation for detailed studies of Ycf1p NBD2 and provides an outline for optimizing the generation of samples of NBD2 from other ABC proteins.

• PMID: 40473008
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Journal Article

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Quail SES, Youn J, Kanelis V

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