Radiolabeling of Mitochondrial Proteins

Radiolabeling of selected mitochondrial proteins (mitochondrial pyruvate carrier 4 (MPC4; At4g22310.1) and Pam18-2 (At3g09700.1)) was performed using well-established procedures [42 (link)]. Briefly, for in vitro transcription, PCR products containing an SP6 promoter upstream the open reading frame were generated using cDNA as a template. Subsequently, in vitro transcription was carried out according to the manufacturer’s recommendation (mMESSAGE mMACHINE SP6 kit; Ambion, Foster City, CA, USA ), RNA was purified (MEGAclear kit; Ambion, Foster City, CA, USA) and used for in vitro translation in reticulocyte lysate (TNT kit; Promega, Mannheim, Germany) that was supplemented with [³⁵S] methionine.