The ubiquinol cytochrome c oxidoreductase complex of spinach leaf mitochondria is involved in both respiration and protein processing

Abstract

Nuclear encoded mitochondrial precursor proteins are cleaved to mature size products by the general mitochondrial processing peptidase (MPP). In contrast to non-plant tissues where MPP is located in the matrix, the general processing activity of potato tuber (storage tissue) mitochondria has been shown to constitute an integral part of the isolated cytochrome c reductase complex of the respiratory chain. Here we show isolation of MPP from photosynthetic tissue, spinach leaf mitochondria, starting from the total membrane processing extract using extraction with dodecyl-β-maltoside followed by FPLC anion-exchange and gel filtration chromatography. The total spinach leaf MPP is found in the fractions containing the cytochrome c reductase complex and is shown to be an integral part of the complex. No processing activity has been found in any other fractions. The isolated cytochrome c reductase complex is shown to process three precursor proteins of different intramitochondrial localisation, the F 1β subunit of ATP synthase (extrinsic membrane protein on matrix side), the Rieske FeS protein (integral membrane protein facing intermembrane space) and the malate dehydrogenase (matrix protein). The processing activity is totally inhibited by EDTA and orthophenanthroline. Our results together with the results in potato mitochondria show that integration of MPP into the cytochrome c reductase is a general phenomenon for plants. The complex consists of ten protein bands on SDS-PAGE of 61, 54, 52, 34, 32, 26, 15, 12, 11 and 10 kDa. The 61, 54 and 52 kDa bands correspond to Core proteins, the 32 kDa band to cytochrome b and the 26 kDa band to Rieske FeS protein as estimated by immunological methods. The three Core proteins are shown to be immunologically related to MPP from other sources, the Core 1 protein corresponding to β-MPP and the Core 2 and Core 3 proteins corresponding to α-MPP, which in comparison to MPP in potato mitochondria indicates species-dependent differences in the appearence of the processing components. Furthermore, the processing activity of the isolated and membrane-bound spinach cytochrome c reductase complex is shown to be inhibited by antimycin A and myxothiazol, electron transfer inhibitors of the complex. The inhibition of processing is, however, not correlated to the inhibition of electron transfer through the complex or to the redox state of the complex.