The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.

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Sample size Sample size calculations were not performed. The amount of protein used was based on protein used in field earlier for ATPase assay and found sufficient based on assay data obtained. All the ATP ase assays were performed at least in technical triplicate and mentioned in figure legends Data exclusions All raw cryo-EM data were processed through the Relion3.0 and Relion3.l pipelines. Micrographs were excluded based on poor resolution Replication Randomization Blinding estimates using CTFFIND which often correlated with heavy ice contamination. Particle selection was performed following 2D and 3D classification in Relion and judging by predicted resolution and visual inspection of classes.
All experiments were performed at least in triplicate (technical replicates) and information provided in figure legends. All attempts were successful and gave consistent results.
Particles were randomized into two half sets following the standard automated Relion protocol.
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