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GENETICS, GENOMICS, AND MOLECULAR EVOLUTION

A Transcriptomic and Proteomic Characterization of the Arabidopsis Mitochondrial Protein Import Apparatus and Its Response to Mitochondrial Dysfunction^1,[w]

Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia

Mitochondria import hundreds of cytosolically synthesized proteins via the mitochondrial protein import apparatus. Expression analysis in various organs of 19 components of the Arabidopsis mitochondrial protein import apparatus encoded by 31 genes showed that although many were present in small multigene families, often only one member was prominently expressed. This was supported by comparison of real-time reverse transcriptase-polymerase chain reaction and microarray experimental data with expressed sequence tag numbers and massive parallel signature sequence data. Mass spectrometric analysis of purified mitochondria identified 17 import components, their mitochondrial sub-compartment, and verified the presence of TIM8, TIM13, TIM17, TIM23, TIM44, TIM50, and METAXIN proteins for the first time, to our knowledge. Mass spectrometry-detected isoforms correlated with the most abundant gene transcript measured by expression data. Treatment of Arabidopsis cell culture with mitochondrial electron transport chain inhibitors rotenone and antimycin A resulted in a significant increase in transcript levels of import components, with a greater increase observed for the minor isoforms. The increase was observed 12 h after treatment, indicating that it was likely a secondary response. Microarray analysis of rotenone-treated cells indicated the up-regulation of gene sets involved in mitochondrial chaperone activity, protein degradation, respiratory chain assembly, and division. The rate of protein import into isolated mitochondria from rotenone-treated cells was halved, even though rotenone had no direct effect on protein import when added to mitochondria isolated from untreated cells. These findings suggest that transcription of import component genes is induced when mitochondrial function is limited and that minor gene isoforms display a greater response than the predominant isoforms.

¹ This work was supported by the Australian Research Council (funds to A.H.M. and J.W. and Australian Postdoctoral Fellowship to A.H.M.), by Australian Postgraduate Awards (to R.L. and R.C.), by University Postgraduate Awards (to O.C. and M.N.L.), and by University of Western Australia (Small Grants Scheme to A.H.M. and J.W.).

^[w] The online version of this article contains Web-only data.

^* Corresponding author; e-mail seamus{at}cyllene.uwa.edu.au; fax 61–08–9380–1148.

Received September 25, 2003; returned for revision October 20, 2003; accepted November 10, 2003.

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