Minisymposium 20: Organelle Development
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M2002: Comparative proteomics of chloroplast protein sorting and proteolysis mutants in Arabidopsis thaliana using differential stable isotope labeling, iTRAQ and cICAT
Protein sorting, folding and degradation are critical in controlling protein homeostasis and cell fitness. Chloroplast cpSRP54 is a conserved member of the Signal Recognition particle and is involved in intra-chloroplast sorting of nuclear- and plastid-encoded integral membrane proteins. The cpSRP54 deletion mutant in Arabidopsis thaliana, assigned ffc, has delayed development and accumulated lower levels of a specific set of membrane proteins. Plant plastids contain tetradecameric Clp protease core complexes, consisting of five serine type protease Clp proteins (ClpP1,3-6), four non-proteolytic ClpR proteins (ClpR1-4) and two associated proteins of unknown function (ClpS1-2). The objective of this comparative proteomics study is to identify the substrates and the functional role(s) of cpSRP54 and the ClpPRS complex in chloroplast development, protein homeostasis and chloroplast metabolism. Using western blotting, native gels and differential stable isotope labeling with cICAT and mass spectrometry of wt and mutant stroma, we show that reduced expression of non-catalytic ClpR2 in Arabidopsis thaliana disrupts chloroplast biogenesis and affect protein homeostasis. Shotgun proteome analyses of young and mature leaves of wt and clpr2 provide an overview in metabolic cellular changes. Different stable isotope labeling with iTRAQ of the digested gel-separated ClpPRS complexes was used to determine possible changes in protein composition in the ClpPRS complex. A quantitative comparison of total cellular proteomes of young wt and ffc seedlings, as well as chloroplasts of mature plants using iTRAQ and cICAT labeling was carried out. A number of striking differences were observed between ffc and wt that could not be attributed to a developmental delay in the ffc mutant.
