In-Depth Analysis of the Thylakoid Membrane Proteome ofArabidopsis thalianaChloroplasts: New Proteins, New Functions, and a Plastid Proteome Database[W]

Abstract

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one-and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were a-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http:// cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

“…Comparison of the 179 proteins identified in this investigation with those from previous proteome investigations of thylakoid 10 and envelope 9 membranes revealed only 17 (9.5%) and 42 (23%) proteins, respectively, in common with those investigations ( Figure 3A). Thylakoid and envelope fractions shared 52 (23%) proteins and all three datasets had 10 proteins in common.…”

Analysis of 101 nuclear transcriptomes reveals 23 distinct regulons and their relationship to metabolism, chromosomal gene distribution and co-ordination of nuclear and plastid gene expression

“…Assignment of subcellular location to the encoded proteins was based on experimental evidence (Kruft et al, 2001;Friso et al, 2004;Heazlewood et al, 2004;Kleffmann et al, 2004) or on stringent computational prediction of subcellular targeting (Richly et al, 2003b;Richly and Leister, 2004). A list of all 1590 genes contained in the 23 regulons, as well as their assignment to functional classes and the subcellular location of the encoded proteins, is provided as Supplementary Material 2.…”

“…Assignment was based on experimental evidence (Kruft et al, 2001;Friso et al, 2004;Heazlewood et al, 2004;Kleffmann et al, 2004) or on stringent computational prediction of subcellular targeting (Richly et al, 2003b;Richly and Leister, 2004). Because the location of only a minority of chloroplast and mitochondrial proteins has been reliably experimentally determined, and because the computational prediction applied was of high specificity but relatively low sensitivity (Richly et al, 2003b;Richly and Leister, 2004), our classification underestimates the number of proteins located in chloroplasts or mitochondria (it follows that in Fig.…”

Regulatory factors for the assembly of thylakoid membrane protein complexes

“…The PSI complexes from RubA mutants lack the stromal subunits PsaC, PsaD and PsaE but contain all of the intrinsic membrane subunits [49]. However, the chloroplast-localized rubredoxin is associated with PSII, but not PSI [50]. Further analysis of the roles of chloroplast rubredoxin is required.…”