Fig. 1: Structure of proteolytically cleaved Ycf1p.

a Schematic diagram of Ycf1p showing the three transmembrane domains (TMD0, TMD1, and TMD2), the L0 linker, the nucleotide binding domains (NBD1, NBD2), the regulatory (R) region, and the coupling helices (CH1, CH2, CH3, and CH4) that are located in TMD1 and TMD2 and contact the NBDs. Phosphorylation sites in the L0 linker (S251^34,121) and R region (S903, S908, T911, and S914^33,35,36) are depicted with a black “P” circled in red. The loop connecting the first two transmembrane helices in TMD1 (loop-6), which contains a 17-residue insertion compared to most other ABCC proteins, is shown by a dashed curve. Cleavage of this loop by Pep4p protease (pink scissors) yields the N-terminal and C-terminal proteolytic fragments⁶⁰. b 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels showing purified samples of cleaved Ycf1p-3xFLAG (left) and uncleaved Ycf1p-3xFLAG (right). The two fragments of cleaved Ycf1p (measuring at ~141 kDa and ~33 kDa from the gel on the left) are consistent with proteolytic cleavage of Ycf1p at the Pep4p site. The migration of the single band in the uncleaved Ycf1p gel is consistent with the 173.8 kDa molecular weight of Ycf1p-3×FLAG. SDS-PAGE gels shown are representative of those for 23 cleaved and 10 uncleaved Ycf1p preparations generated for this study. c Analytical gel filtration chromatography of cleaved (black trace) and uncleaved (grey trace) Ycf1p. Both cleaved and uncleaved Ycf1p samples contain dimeric forms of the protein, with cleaved Ycf1p samples containing up to ~40% dimers. d 2D class average images showing cleaved Ycf1p monomer (left) and dimer (right). e Cryo-EM maps for two different conformations of the cleaved Ycf1p monomer in which the NBDs have different degrees of separation (IFwide-α and IFwide-β). The protein domains are colored as in panel (a) and the lipids are colored in grey. f Cryo-EM map of cleaved dimeric Ycf1p, in which both protomers are in the IFwide-β conformation, with protein domains coloured as in panel (a) and lipids in grey.