Fig. 1. The P5A-ATPase directly interacts with the TM of TA proteins.

(A) Scheme to identify direct interactors of a mitochondrial TA protein. Recombinant FLAG-tagged TA protein with the OMP25 transmembrane segment (TM) containing the UV-reactive crosslinker (photo-XL) Bpa was purified in complex with calmodulin [FLAG-TA(Bpa)-CaM]. EGTA dissociates FLAG-TA(Bpa) from CaM, permitting TM insertion into organelles in a crude membrane fraction or association with purified SGTA. Exposure to ultraviolet (UV) light induces site-specific covalent crosslinks. (B) Anti-FLAG blots of reactions as in (A). Unmodified FLAG-TA(Bpa) and FLAG-TA(Bpa) crosslinks of oligomers (x TA) and to SGTA (x SGTA) or membrane-specific proteins (orange asterisks) are indicated. (C) 6 replicates of reactions as in (A) without or with UV irradiation were affinity purified and analyzed by quantitative multiplexed mass spectrometry with tandem mass tagging (TMT-MS). Volcano plot shows relation of the P-value and log₂ enrichment of proteins by UV irradiation. (D) 5% input (tot.) of reactions as in (A) with organelles containing Spf1-GFP were purified via FLAG-TA(Bpa) (elu.) and immunoblotted for GFP to show UV-dependent Spf1 interaction. (E) Autoradiography of total (tot.) and denaturing anti-FLAG immunoprecipitations (IPs; top) of photocrosslinking reactions of in vitro translated radiolabeled untagged TA(Bpa) with organelles containing Spf1-FLAG. OMP25 TM and C-terminal domain (CTD) sequence is below. Bpa is positioned in the cytosolic (cyto.) domain (lanes 1-2) or the OMP25 TM (lanes 3-5). R-E, TA(Bpa) in which the arginines in the OMP25 CTD are mutated to glutamates; trunc. TA, truncated translation products that do not incorporate Bpa.