Towards an Analysis of the Rice Mitochondrial Proteome

Abstract

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 ϭ Ϫ1.27 to ϩ0.84), highly basic and acid proteins (isoelectric point ϭ 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.The ATP-synthesizing organelles of eukaryotic cells, mitochondria, can trace their origins back to an event in which one prokaryotic cell was engulfed by another cell to form a new cellular lineage containing two genomes. These genomes became dependent over time, and mitochondria lost the ability to be viable outside the host cell. A significant net transfer of genetic information occurred from the mitochondrial to the nuclear genome during this time. It is predicted that mitochondria now synthesize only a few percent of the proteins required for their function, with the majority of their proteins being encoded in the nucleus, translated in the cytosol, and transferred back to the mitochondria as protein precursors by means of targeting information in the protein sequence (Gray et al., 1999). Biochemical investigations have revealed that isolated plant mitochondria shar...

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs

“…Batches of 200 g of rice (Oryza sativa 'Amaroo') seeds were washed in 1% (w/v) bleach for 10 min, rinsed in distilled water, grown in the dark in vermiculite trays (30 3 40 cm) at a constant 30°C, and watered daily, and the shoots were harvested at 10 d for mitochondrial isolation (Heazlewood et al, 2003). Rice shoots were harvested using scissors and cut into 5-to 10-mm lengths into a beaker on ice.…”

Changes in the Mitochondrial Proteome during the Anoxia to Air Transition in Rice Focus around Cytochrome-containing Respiratory Complexes

“…Further, the much reduced state of mitochondria in anoxia in yeast suggest an oxygen-derived trigger for full mitochondrial biogenesis, but the nature of mitochondrial proteomes under anoxia have not been systematically investigated to date in either anoxia-tolerant or -intolerant plants. We have recently studied the rice mitochondrial proteome in air-grown seedlings (25). By following the induction of respiration in rice coleoptiles during air adaptation of anoxic seedlings and assessing the changes in the rice mitochondrial proteome during this transition, we showed that much of the mitochondrial proteome appears relatively unaffected by anaerobic growth.…”

Update and challenges on proteomics in rice

“…In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. Heazlewood et al [67] also reported the first direct identification, to our knowledge, of pentatricopeptide repeat proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture of mitochondrial functions in a model monocot plant.…”

“…This technique allows the separation in two dimensions of extremely hydrophobic protein sets and provides information on their native interactions [65]. Heazlewood et al (2003) reported rice mitochondrial proteome analysis using BN-PAGE. Purified rice mitochondrial proteins have been arrayed by IEF-PAGE, by BN-PAGE, and by RP HPLC separation.…”

“…Purified rice mitochondrial proteins have been arrayed by IEF-PAGE, by BN-PAGE, and by RP HPLC separation. From these arrays, Heazlewood et al [67] identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins, highly basic and acidic proteins, and proteins over a large molecular mass range, using proteomic approaches. BN-PAGE has provided a detailed picture of electron transport chain protein complexes.…”