The proteome of higher plant mitochondria.

Plant mitochondria perform a wide range of functions in the plant cell ranging from providing energy and metabolic intermediates, via coenzyme biosynthesis and their own biogenesis to retrograde signaling and programmed cell death. To perform these functions, they contain a proteome of >2000 different proteins expressed in some cells under some conditions. The vast majority of these proteins are imported, in many cases by a dedicated protein import machinery. Recent proteomic studies have identified about 1000 different proteins in both Arabidopsis and potato mitochondria, but even for energy-related proteins, the most well-studied functional protein group in mitochondria, <75% of the proteins are recognized as mitochondrial by even one of six of the most widely used prediction algorithms. The mitochondrial proteomes contain proteins representing a wide range of different functions. Some protein groups, like energy-related proteins, membrane transporters, and de novo fatty acid synthesis, appear to be well covered by the proteome, while others like RNA metabolism appear to be poorly covered possibly because of low abundance. The proteomic studies have improved our understanding of basic mitochondrial functions, have led to the discovery of new mitochondrial metabolic pathways and are helping us towards appreciating the dynamic role of the mitochondria in the responses of the plant cell to biotic and abiotic stress.

Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.