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

Nitesh Kumar Khandelwal,Cinthia R Millan,Samantha Avila,Dewight Williams,Thomas M Tomasiak,Samantha I Zangari,Tarjani M Thaker

doi:10.1038/s41467-022-28811-w

Abstract

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.

Highlights

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress
To enable structural studies of Ycf[1], we expressed and purified two forms in the S. cerevisiae DSY5 strain: wild-type (WT) and the NBD2 Walker B mutant E1435Q (Supplementary Fig. 1). Grids of both WT and E1435Q Ycf[1] in apo conditions were prepared as described in the methods, but only the E1435Q Ycf[1] dataset yielded homogenous particles from which we successfully identified not one but two inward-facing conformations determined to 3.4 and 4.0 Å resolution (Fig. 1b, c)
The E1435Q mutant resulted in substantially lowered ATPase activity compared to WT Ycf[1] (Vmax 95 ± 2 nmol/min/mg) (Fig. 1d), likely allowing the trapping of these conformations

Summary

Introduction

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Performs such a protective function by transporting the tripeptide glutathione, the main non-protein thiol in cells, upon oxidation to maintain redox balance or after conjugation to toxic heavy metals such as cadmium, mercury, or lead into vacuoles[1–5]. These metals make up three of the top ten environmental toxins as defined by the CDC Ycf[1] acts as a major redox sink and regulates redox levels in cytoplasm by sequestering glutathione after it is oxidized In this way, Ycf[1] serves a powerful protective function against metals and ROS, both of which are electrophiles that attack DNA, proteins, and the cell membrane.

Methods

Results

Conclusion