Molecular structure of the 8.0 kDa subunit of cytochrome-c reductase from potato and its Δψ-dependent import into isolated mitochondria

AbstractThe cytochrome-c reductase (EC 1.10.2.2) of the mitochondrial respiratory chain couples electron transport from ubiquinol to cytochrome c with proton translocation across the inner mitochondrial membrane. The enzyme from potato was shown to be composed of 10 subunits. Isolation and characterization of cDNA clones for the second smallest subunit reveal an open reading frame of 216 bp encoding a protein of 8.0 kDa. The protein exhibits similarities to a 7.2/7.3 kDa subunit of cytochrome-c reductase from bovine and yeast, that is localized on the intermembrane space side of the enzyme complex. It also shows similarity to a previously unidentified 7.8 kDa protein of cytochrome-c reductase from Euglena. The potato 8.0 kDa protein has a segmental structure, as its sequence can be devided into four parts, each comprising a central Arg-(Xaa)5-Val motif. N-terminal sequencing of the mature 8.0 kDa protein indicates the absence of a cleavable mitochondrial targeting sequence. Import of the in vitro synthesized 8.0 kDa protein into isolated potato mitochondria confirms the lack of a presequence and reveals a dependence of the transport on the membrane potatial Δψ across the inner mitochondrial membrane. These features are unique among the intermembrane space proteins known so far

The alternative Medicago truncatula defense proteome of ROS - defective transgenic roots during early microbial infection

ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homolog in plants). Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i) infected with pathogenic (Aphanomyces euteiches) and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti). While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector), we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi), 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique) were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique) after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique) after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein degradation

New insights into the co-evolution of cytochrome c reductase and the mitochondrial processing peptidase

The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome c reductase complex from higher plants. Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochrome c reductase complex. To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity. Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algae Polytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution. This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome c reductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity. An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochrome c reductase complex and the occurrence of chloroplasts

Blue native DIGE as a tool for comparative analyses of protein complexes

Differential gel electrophoresis (DIGE) is based on pre-labeling of different protein fractions and their subsequent co-electrophoresis in a single gel. Cyanine based "CyDye DIGE Fluor minimal dyes" are used for the labeling reaction and 2D IEF/SDS PAGE is the preferential electrophoresis system for protein separation. The DIGE technology allows elimination of inconsistencies based on gel to gel variations and furthermore allows exact quantification of proteins separated by gel electrophoresis. Here we report applications of the DIGE technology in combination with another 2D gel system, Blue native/SDS PAGE. "Blue native DIGE" offers (i) systematic and quantitative comparison of protein complexes of related protein fractions, (ii) structural investigation of protein complexes, (iii) assignment of protein complexes to subcellular fractions like organelles and (iv) electrophoretic mapping of isoforms of subunits of protein complexes with respect to a larger proteome. The potential of "Blue native DIGE" is illustrated by analysis of organellar fractions from the plant Arabidopsis thaliana and the alga Polytomella. Use of the DIGE technology for topological investigations is discussed

Unique composition of the preprotein translocase of the outer mitochondrial membrane from plants

Transport of most nuclear encoded mitochondrial proteins into mitochondria is mediated by heteropolymeric translocases in the membranes of the organelles. The translocase of the outer mitochondrial membrane (TOM) was characterized in fungi, and it was shown that TOM from yeast comprises nine different subunits. This publication is the first report on the preparation of the TOM complex from plant mitochondria. The protein complex from potato was purified by (a) blue native polyacrylamide gel electrophoresis and (b) by immunoaffinity chromatography. On blue native gels, the potato TOM complex runs close to cytochrome c oxidase at 230 kDa and hence only comprises about half of the size of fungal TOM complexes. Analysis of the TOM complex from potato by SDS-polyacrylamide gel electrophoresis allows separation of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa. The 23-kDa protein is identical to the previously characterized potato TOM20 receptor, as shown by in vitro assembly of this protein into the 230kDa complex, by immunoblotting and by direct protein sequencing. Partial amino acid sequence data of the other subunits allowed us to identify sequence similarity between the 36-kDa protein and fungal TOM40. Sequence analysis of cDNAs encoding the 7-kDa protein revealed significant sequence hornology of this protein to TOM7 from yeast. However, potato TOM7 has a N-terminal extension, which is very rich in basic amino acids. Counterparts to the TOM22 and TOM37 proteins from yeast seem to be absent in the potato TOM complex, whereas an additional low molecular mass subunit occurs. Functional implications of these findings are discussed

Uniform nomenclature for the protein transport machinery of the mitochondrial membranes

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