Analysis of curated and predicted plastid subproteomes of Arabidopsis. Subcellular compartmentalization leads to distinctive proteome properties

Abstract

Carefully curated proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These curated proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional (low abundant) components of each membrane system. The assembly of rigorously curated subcellular proteomes is in itself also important as a parts list for plant and systems biology. Transmembrane and subcellular prediction strategies were evaluated using the curated data sets. The three curated proteomes differ strongly in average isoelectric point and protein size, as well as transmembrane distribution. Removal of the cleavable, N-terminal transit peptide sequences greatly affected isoelectric point and size distribution. Unexpectedly, the Cys content was much lower for the thylakoid proteomes than for the inner envelope. This likely relates to the role of the thylakoid membrane in light-driven electron transport and helps to avoid unwanted oxidation-reduction reactions. A rule of thumb for discriminating between the predicted integral inner envelope membrane and integral thylakoid membrane proteins is suggested. Using a combination of predictors and experimentally derived parameters, four plastid subproteomes were predicted from the fully annotated Arabidopsis genome. These predicted subproteomes were analyzed for their properties and compared to the curated proteomes. The sensitivity and accuracy of the prediction strategies are discussed. Data can be extracted from the new plastid proteome database (http://ppdb.tc.cornell.edu).

Figure 1.

Frequency distribution of predicted TMDs of the curated membrane subproteomes. The curated sets are the extended nuclear-encoded (n) thylakoid membrane proteome with 83 proteins (A–C), the extended inner envelope membrane proteome with 39 proteins (A and B), and the chloroplast-encoded (c) thylakoid membrane proteome with 38 members (A). TMD predictions were by TMHMM (A–C) or consensus prediction as reported in Aramemnon (B and C). Predicted transit peptides were removed. All accession numbers are listed in Supplemental Table I.

Figure 2.

Hydrophobicity and TMD prediction of the curated membrane proteomes. Correlation of the ratio between predicted number of TMDs and the number of amino acid residues (TMD:aa) and GRAVY index for the expanded nuclear-encoded inner membrane envelope (A) and the expanded thylakoid membrane proteomes (B), as well as the chloroplast-encoded thylakoid membrane proteome (C). These are the same sets as used for Figure 1. TMDs were obtained from Aramemnon (A and B) or predicted by TMHMM (C). The regression analysis is indicated for each plot.

Figure 3.

pI and Cys content of the expanded curated lumenal, thylakoid membrane and inner envelope membrane proteome. A, Frequency distribution of the pI for the full-length and processed curated proteomes (full-length, white squares, cTP removed, black squares, and cTP and lTP removed, black triangles). B, Frequency distribution of proteins in the three curated proteomes based on the number of Cys residues. Membrane sets are the same as used in Figures 1 and 2, and the lumenal set contained 51 proteins (see Supplemental Table I).

Figure 4.

Overview of the two prediction strategies and comparisons of predictions and experimentation. A, Prediction strategy using LumenP. B, Prediction strategy using SignalP. C, Overlap of proteins predicted by the two prediction strategies with 291 and 285 proteins, respectively, and the experimental lumenal proteome with 53 proteins (using all gene models in TAIR). D, Comparison of distribution cTP + lTP length for the curated and predicted lumenal proteomes.

Figure 5.

Scatter plot of the number of Cys residues, protein length, and pI for the curated sets of inner envelope and thylakoid membrane proteins. Predicted cTPs are removed. A, 83 thylakoid and 39 envelope membrane proteins. B, 110 and 61 thylakoid and envelope membrane proteins, respectively. Envelope proteins are symbolized by blue dots and thylakoid proteins by red dots.