Functional proteomics of chloroplast nucleoproteins towards an understanding of nucleoid structure, function and dynamics

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Chloroplast nucleoids contain a set of DNA-binding proteins some of which have architectural function whereas others are likely to act as key regulators of gene expression. We propose to harness the enormous genetic potential of the model organism Chlamydomonas reinhardtii (also referred to as "green yeast") in order to address key questions of chloroplast nucleoid structure and function. The proposed research involves the large-scale purification of chloroplast nucleoids from Chlamydomonas and their systematic proteomics analysis with the ultimate goal to identify the complete set of DNA-binding proteins (structural nucleoid proteins and regulator proteins) associated with the chloroplast genome. We aim to characterize a subset of these factors molecularly and, in addition, we will explore the dynamics of nucleoid architecture by comparatively analyzing the set of DNA-binding proteins associated with chloroplast nucleoids in various environmental conditions and developmental stages. This comparative functional proteomics approach will provide new insights into the regulation of higher-order structures of the chloroplast DNA and the control of chloroplast gene expression through DNA-binding regulator proteins in response to changing environmental conditions and developmental programs. Opinion of the review panel (including one written comment): Prof. Bock wants to isolate nucleoids from Chlamydomonas chloroplasts and identify the proteins contained in these structures. The proteins shall then be checked as GFP fusions for their presumed localisation within the nucleoids. The final aim of this project is to analyse the function of nucleoid proteins by means of the RNAi technology and, furthermore, by comparing the protein composition of nucleoids under different conditions. Prof. Bock is well experienced in genetics and molecular biology of higher plant chloroplasts and has an excellent publication record in this field. The project outlined in the original written proposal was considered very interesting and timely because little is yet known about chloroplast nucleoid proteins and because of the expected impact on understanding prokaryotic nucleoids. However, the reviewers were worried that the set-up was too broad to allow the project to proceed much beyond cataloguing of a large number of candidate proteins. They, therefore, requested that clear selection criteria be given for proteins to be characterized further on a functional level, and thereby the project be more strictly focussed. In addition, a few technical details were asked to be clarified.