In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes

Abstract

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx(5)HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.

Figure 1.

Purity analysis and 1-D gel separation of peroxisomal proteins. A and B, Leaf peroxisomal proteins (5 μg each lane) were separated on acrylamide minigels. Anti-VDAC immunoblotting (A) and silver staining (B) served to determine the relative contents of mitochondrial VDAC and the P-protein, respectively, while verifying the total amount of loaded proteins in parallel (B). According to the relative content of mitochondrial proteins, leaf peroxisome isolates were classified as of low (lane 1), moderate (lane 2), and high (lane 3) purity. All lanes in each panel are from the same gel. C, Leaf peroxisomal proteins (500 μg) of highest organelle purity were separated on a 10% SDS-PAGE minigel. After brief staining, the gel was cut into 16 slices and proteins were in-gel digested with trypsin and analyzed by LC-MS/MS. The line in S2 separates the stacking and resolving gels.

Figure 2.

Subcellular and functional classifications of proteins identified in leaf peroxisomes. A, Number of peroxisomal and nonperoxisomal proteins grouped by NSAF values, which correlate with protein abundance. B, Relative abundance and protein numbers of peroxisome-associated and nonperoxisomal proteins. C, Functional assignment of peroxisome-associated proteins shown by relative abundance and protein numbers.

Figure 3.

Analysis of PTS conservation in homologous plant ESTs for putative novel proteins. Asterisks indicate stop codons. Plant species abbreviations are defined in Supplemental Document S1.

Figure 4.

Peroxisomal localization of 12 proteins containing predicted PTSs. Confocal microscopic images were taken from leaf epidermal cells of 4-week-old tobacco plants in which YFP fusions and the CFP-PTS1 peroxisomal marker were coexpressed. Bars = 10 μm.

Figure 5.

Identification of two novel PTS1 tripeptides, SKV> and SLM>. Confocal images were obtained from leaf epidermal cells of 4-week-old tobacco plants coexpressing the indicated YFP fusions and the CFP-PTS1 peroxisomal marker. Bars = 10 μm.

Figure 6.

Dual localization of the HIT3 protein, which has an N-terminal PTS2-related sequence. Confocal images taken from leaf epidermal cells of 4-week-old tobacco plants coexpressing the indicated YFP fusion and the CFP-PTS1 peroxisomal marker are shown. Bars = 10 μm.

Figure 7.

Peroxisomal localization of four novel proteins without predicted PTSs. Shown are confocal images taken from leaf epidermal cells of 4-week-old tobacco plants coexpressing the indicated YFP fusion proteins and CFP-PTS1. Bars = 10 μm.

Figure 8.

Comparison of Arabidopsis peroxisomal proteome data. The number of peroxisomal proteins identified in this study was compared with that from previous proteome studies by Reumann et al. (2007), Fukao et al. (2002, 2003), and Eubel et al. (2008). One protein is shared uniquely by Reumann et al. (2007) and Eubel et al. (2008) and could not be displayed on this Venn diagram.