Prototeomics of the Thylakoids from Arabidopsis Thaliana; Protein Identification and Interactions, Post-Translational Modifications and Differential Expression

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The chloroplasts in green algae and higher plants are predicted to contain around 2500-3000 different proteins, corresponding to 10-12% of the total plant proteome. Chloroplasts possess thylakoid membranes with four multi-subunit protein complexes, comprising together at least 66 different proteins and carrying out the photosynthetic reactions. Assembly of these complexes involves targeting, insertion, processing as well as ligation of cofactors, and requires regulation at transcriptional, translational and post-translational levels. At least 100 proteins involved in such biogenesis related processes should be present in the thylakoid membrane, but only a few have been identified so far. With the completion of the Arabidopsis thaliana genome and the improvement of 2-dimensional electrophoresis and mass spectrometry (MS), a systematic characterization of the thylakoid proteome has become feasible. The objective of this project is to systematically characterize the thylakoid proteome of Arabidopsis thaliana. This characterization includes a global identification of most thylakoid proteins and their protein-protein interactions. Highly reproducible 2-dimensional electrophoresis, non-denaturing biochemical separation techniques, and state of the art mass spectrometers will be employed to obtain this characterization in a time-effective manner. On-line MS will be carried out for the notoriously difficult identification of small proteins or very hydrophobic integral membrane proteins. Together this will give a unique overview of the thylakoid proteome. In addition, it will also enable more rational design (reverse) genetic strategies for studies of thylakoid biogenesis and maintenance. A successful realization of the project could serve also to deepen our general understanding of organelle biogenesis, as well as inner membrane biogenesis of prokaryotes. In summary, the objectives of this project are:I. Systematic identification of soluble and peripheral thylakoid proteins by mass spectrometry from gel-separated proteins from 1-dimensional and 2-dimensional gels. II. Identification of integral thylakoid membrane proteins by fractionation in organic solvents and on-line tandem mass spectrometry (reverse phase HPLC). III. Selected identification of (low abundant) thylakoid protein complexes and protein-protein interactions by non-denaturing purification methods and mass spectrometry. IV. Analysis of chloroplast targeting signals and feedback to prediction programs and analysis of putative discrepancies between predicted protein sequences (from cDNA or genomic DNA) and experimental determined protein sequences. V. Development of a professional custom built proteomics LIM (laboratory information management) system, including a web interface.