Genomic and Proteomic Analysis of Mitochondrial Carrier Proteins in Arabidopsis

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Abstract
Plant mitochondria maintain metabolic communication with the cytosol through a family of carrier proteins. In Arabidopsis, a subset of 45 putative genes encoding members of this family have been identified based on generalized mitochondrial carrier features. No gene clusters are apparent and few of the predicted protein products have mitochondrial targeting sequences recognized by bioinformatic predictors. Only nine genes are currently represented by more than 10 expressed sequence tags at The Institute for Genomic Research. Analyses of public microarray experiments reveal differential expression profiles of the more highly expressed members of this gene family in different plant organs and in response to plant hormone application and environmental stresses. A comparison of this Arabidopsis carrier subset (45) to the yeast gene family (35) reveals 10 orthologous groups between the two species. Recent surveys of the Arabidopsis mitochondrial proteome by two-dimensional gel separations have not identified any of these carrier proteins, presumably because of their hydrophobicity and basicity. Isolating integral membrane proteins from Arabidopsis mitochondria, using one-dimensional electrophoresis for protein separation and tandem mass spectrometry-based sequencing of doubly charged peptides, we have unequivocally identified specific carrier gene products located in mitochondria. This approach has identified six of the nine carriers represented highly in expressed sequence tag databases: adenine nucleotide translocator (At3g8580 and At5g13490), dicarboxylate/tricarboxylate carrier (At5g19760), phosphate carrier (At5g14040), uncoupling protein (At3g54110), and a carrier gene of unknown function (At4g01100). Overall, the combined transcript and protein expression data indicates that only a small subset of the carrier family of genes provide the majority of carrier proteins of Arabidopsis mitochondria.