The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins

Abstract

Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.

Figure 1.

Schematic of Proteomic Studies Conducted on the Vegetative Vacuole. (A) Purified vacuoles (bar = 50 μm) were subjected to (1) in-liquid trypsin digestion followed by 2-D LC MS/MS or (2) 1-D SDS-PAGE followed by in-gel digestion and LC MS/MS, or (3) tonoplast fractions were first enriched and then subjected to 1-D SDS-PAGE followed by in-gel digestion and LC MS/MS. (B) Mass spectrometry output from LC MS/MS. The fragmentation spectrum (MS/MS) of a peptide derived from a low-abundance tonoplast SNARE protein, SYP22 (At5g46860), is shown. The precursor ion was doubly charged with a mass-to-charge ratio (m/z) 1120.17. The spectrum was matched by MASCOT database searching to a peptide, EQGIQEIHQQIGEVNEIFK (amino acids 182 to 200), of SYP22. All matched y- and b-series ions are labeled. Amino acid residues are assigned based on the mass ladders generated by the b-series ions.

Figure 2.

Distribution of Identified Proteins by Different Methods. Overlap of the different protein sets is shown. Numbers in parentheses indicate the total number of proteins found by a particular method.

Figure 3.

Bioinformatic Analyses of the Identified Proteins. (A) Size distribution of the identified proteins in 10-kD increments. (B) Distribution of proteins versus isoelectric point (pI). (C) Number of predicted transmembrane domains identified in vacuolar proteins as determined by the HMMTOP method. Note that all proteins with a predicted signal peptide and a single predicted transmembrane domain were removed before this analysis because the transmembrane domain prediction programs used usually identify signal peptides as a transmembrane domain.

Figure 4.

Schematic of the Plant Vacuole and Functional Categorization of the Identified Vacuolar Proteins. Identified proteins were grouped into eight major categories, and the number of proteins belonging to each group are shown in parentheses. Some of the specific activities of each group and a model of the tonoplast SNARE-pin complex are also shown.

Figure 5.

Proteins with Putative Vacuolar Sorting Determinants. (A) Alignment of the C-terminal region of vacuolar peroxidases with closely related peroxidases from Arabidopsis. All identified vacuolar peroxidases (labeled with an asterisk) have a hydrophobic C-terminal extension that may serve as a CTPP sorting signal. Only three peroxidases in the Arabidopsis genome, At2g38380, At2g38390 (data not shown), and At5g06730, have a similar C-terminal extension but were not found in the vegetative vacuole. Horseradish peroxidase C1a is also included in the alignment (GenBank accession no. P00433), and the native site of peptide cleavage is indicated with an arrowhead. (B) Aligned regions of putative NTPP-containing proteins. Proteins containing the degenerate signal [N/L]-[P/I/L]-[I/P]-[R/N/S] immediately following the predicted signal peptide cleavage site are shown, and the putative NTPP signals are in bold and underlined.