Identification of the plant mitochondrial OrfX protein: A mass spectrometry approach.

The mitochondrial genome of plants contains an open reading frame, orfx, which encodes a protein classified as very rare and which has so far escaped mass spectrometric detection. The protein resembles the c-subunit of bacterial twin-arginine-motif-dependent protein translocases (TatC). Using native prefractionation of mitochondrial protein complexes from Arabidopsis and trapped-ion mobility spectrometry (TIMS) time-of-flight mass spectrometry, we report the identification of three peptides of the orfx protein. Our experimental approach was used to trace the native forms of the protein and show that it is present in protein complexes in the size range of 450-530 kDa, which also contain TatB. We suggest that mitochondrial TatBC complexes in plants may bind to late assembly intermediates of respiratory chain complex III.