RESEARCH ARTICLE A survey of the Arabidopsis thaliana mitochondrial phosphoproteome Jun Ito 1,2 , Nicolas L. Taylor 1 , Ian Castleden 3 , Wolfram Weckwerth 4 , A. Harvey Millar 1,2 and Joshua L. Heazlewood 1,2,4 1 Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Western Australia, Australia 2 School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia 3 Centre of Excellence for Computational Systems Biology, The University of Western Australia, Crawley, Western Australia, Australia 4 Max-Planck Institute for Molecular Plant Physiology, Potsdam-Golm, Germany Received: February 2, 2009 Revised: June 8, 2009 Accepted: June 16, 2009 Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabi- dopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phospho- proteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2-DE separations of Arabidopsis mitochon- drial proteins and affinity-enriched phosphoproteins using the Pro-Q Diamond phospho- specific in-gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphor- ylation at both the subcellular and the cellular level in Arabidopsis. Keywords: Arabidopsis / Mitochondria / Phosphoproteome / Phosphorylation/ Plant proteomics 1 Introduction Protein phosphorylation is a major regulatory mechanism in controlling various cellular processes and it is estimated that up to one-third of all eukaryotic proteins are phos- phorylated at any given time [1, 2]. The first report of protein phosphorylation in plant mitochondria was of pyruvate dehydrogenase complex (PDC) of cauliflower, which was inactivated and phosphorylated by pyruvate dehydrogenase kinase [3]. More extensive investigations of plant mitochondrial protein phosphorylation using 1-D PAGE and Abbreviations: E1a, E1-alpha subunit of PDC; FDH, formate dehydrogenase; GDH, glutamate dehydrogenase; MAM, mito- chondrial acid matrix; MOAC, metal oxide affinity chromatogra- phy; PDC, pyruvate dehydrogenase complex; SDH, succinate dehydrogenase; TiO 2 , titanium dioxide Correspondence: Dr. Joshua L. Heazlewood, Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 1 Cyclotron Road MS 978-4466, Berkeley, CA 94720-8205, USA E-mail: jlheazlewood@lbl.gov Fax:11-510-486-4252 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2009, 9, 4229–4240 4229 DOI 10.1002/pmic.200900064