First published online April 10, 2003; 10.1104/pp.102.018986
Plant Physiol, May 2003, Vol. 132, pp. 230-242
Towards an Analysis of the Rice Mitochondrial
Proteome1
Joshua L.
Heazlewood,
Katharine A.
Howell,
James
Whelan, and
A. Harvey
Millar*
Plant Molecular Biology Group, School of Biomedical and Chemical
Sciences, The University of Western Australia, Crawley 6009, Western
Australia, Australia
Purified rice (Oryza sativa)
mitochondrial proteins have been arrayed by isoelectric
focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN)
PAGE, and by reverse-phase high-performance liquid chromatography (LC)
separation (LC-mass spectrometry [MS]). From these protein arrays, we
have identified a range of rice mitochondrial proteins, including
hydrophilic/hydrophobic proteins (grand average of hydropathicity =  1.27 to +0.84), highly basic and acid proteins (isoelectric
point = 4.0-12.5), and proteins over a large molecular mass range
(6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed
picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations
were excised, trypsin digested, and analyzed by tandem MS (MS/MS).
Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open
reading frames from genomic sequence and six-frame translated rice
expressed sequence tags. Sequence comparison allowed us to assign
functions to a subset of 85 proteins, including many of the major
function categories expected for this organelle. A further six spots
were matched to rice sequences for which no specific function has yet
been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence
entries from open reading frame and expressed sequence tag databases.
Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In
total, 136 nonredundant rice proteins were identified, including a new
set of 23 proteins of unknown function located in plant mitochondria.
We also report the first direct identification, to our knowledge, of
PPR (pentatricopeptide repeat) proteins in the plant mitochondrial
proteome. This dataset provides the first extensive picture, to our
knowledge, of mitochondrial functions in a model monocot plant.
1
This work was supported by the Australian
Research Council Discovery Program (to A.H.M. and J.W.). K.A.H. was a
recipient of the Eric Cyril Lawrence Medical Research Scholarship.
*
Corresponding author; e-mail hmillar{at}cyllene.uwa.edu.au; fax
61-8-9380-7245.
© 2003 American Society of Plant Biologists
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