Purification of Mitochondria from Arabidopsis Cell Cultures.
(A) Marker enzyme distribution in first and second density gradient separations of crude organelle pellets to yield purified mitochondria. Fractions 1 to 23 are 1.5-mL fractions from top to bottom of the gradients. Alkaline pyrophosphatase (PPase) is used as a plastid marker, cytochrome c oxidase (COX) is used as a mitochondrial marker, catalase (CAT) is used as a peroxisome marker, and alcohol dehydrogenase (ADH) is used as a cytosolic marker. Activities in each fraction are reported as percentages of activity found in crude organelle pellets.
(B) Specific activities of marker enzymes in the mitochondria-containing fractions at different stages of the purification process (means ± sd, n = 4). The crude organelle pellet protein is ∼60% plastidic, ∼30% mitochondrial, and ∼10% peroxisomal. Based on these proportions and the specific activities shown here, the final mitochondrial fractions contain ∼1.5% protein of plastidic origin and ∼0.2% protein of peroxisomal origin. n.d., not determined.

Low-Abundance Proteins Identified by LC-MS/MS.
Comparisons of the number of ESTs for each protein between the total gel-based proteomic analysis (180), the total LC-MS/MS set of proteins (390), and the novel proteins identified only by LC-MS/MS (236). EST numbers were derived from TIGR annotation data. All data are presented as numbers of nonredundant proteins in the EST number ranges indicated.

Major Functional Distributions of the Arabidopsis Mitochondrial Set and of Matching Putative Orthologs from Yeast, Human, and Rickettsia.
Orthologs were determined through high-stringency sequence homology matching using the program INPARANOID (Remm et al., 2001). The distribution of putative orthologs to the reduced Arabidopsis mitochondrial set of 377 proteins is presented for the three major functional classes of energy (E; white), metabolism (M; light gray), and protein fate (P; dark gray) and for all other identifications (O; black). The total number of putative orthologs in each species, in at least one set, and in all three sets is shown above each bar.

Evaluation of Mitochondrial Prediction in Arabidopsis by Intersections between Targeting Prediction Programs.
(A) Venn diagram illustrating the overlap of mitochondrial prediction of the Arabidopsis nucleus-encoded proteome comprising 27,288 proteins (TIGR ATH1 release 3.0 including supplementary data). The subcellular prediction programs MitoProt II, iPSORT, Predotar, and TargetP were chosen for whole-proteome analysis because they all displayed similar sensitivities with regard to mitochondrial localization (Table 2). The number of mitochondria-targeted proteins predicted by all four programs is underlined (white central section); the six other white areas indicate two-way predictive relationships between the programs. The inner black regions indicate three-way predictions, and the outer black areas indicate the number of novel proteins predicted by each program. Numbers in parentheses represent the total number of mitochondrial proteins predicted by each program. The number outside the Venn diagram (18,468) represents the null set.
(B) Relational interaction between averaged sensitivities using multiple predictors with the experimentally derived nonredundant set of 368 nucleus-encoded proteins from Arabidopsis. The use of multiple predictions in the assessment of mitochondrial localization results in a substantial reduction in sensitivity. Standard deviations are presented as error bars: one predictor, n = 4; two predictors, n = 6; three predictors, n = 4; four predictors, n = 1.