Figure 1.

Purification of Shy1 protein complexes. (A) Shy1^Prot.A construct used for isolation of Shy1 complexes. Gray box, mature Shy1 protein; black boxes, predicted transmembrane domains (TM); PS, predicted cleavable presequence; Prot.A, Protein A tag; TEV, TEV cleavage site; 7His, heptahistidine tag. (B) Growth test on fermentable and non-fermentable medium. Cells were spotted in serial 10-fold dilutions and incubated at 30°C. WT, wild-type. (C) Mitochondria from wild-type (WT) and Shy1^Prot.A were solubilized in 1% digitonin buffer and subjected to IgG chromatography. After TEV-protease cleavage, eluates were separated on tricine-SDS–PAGE, stained with Colloidal Coomassie, or analyzed by Western blotting. Bands were excised and subjected to mass spectrometric analysis. Asterisks denote degradation products identified by mass spectrometry. (D) Eluates from panel C were analyzed by SDS–PAGE and immunodecoration. Total, 1%; eluate, 100%. (E) Cox14^TAP and Mss51^TAP complexes were isolated from digitonin-solubilized mitochondria via IgG chromatography, proteins separated by SDS–PAGE, and analyzed by Western blotting. Total, 2%; eluate 100%. F₁β, β subunit of F₁F_oATP synthase.