Mass Spectrometry-Based Characterization Of Chloroplast Protein Complexes And Clp Protease Function

Mass spectrometry (MS) has enabled large-scale protein identification and quantification, yielding significant insights into relevant biological systems. MS has been extensively applied in this thesis to study chloroplast protein complexes and to quantify protein expression levels in the model plant Arabidopsis thaliana. Plant plastids contain a 350 kDa Clp protease core complex consisting of two heptameric rings. The complex contains nine different proteins in one or more copies, namely five serine-type ClpP peptidases (ClpP1,3-6) and four non-proteolytic ClpR subunits (ClpR1-4). This core complex was purified from different transgenic lines harboring affinity-tagged Clp subunits. The absolute amount of each subunit and the corresponding stoichiometry within the heptameric rings was determined by MS analysis using stable isotope-labeled versions of peptides that uniquely represent each Clp protein, expressed from a synthetic gene. Results showed that the ClpP and ClpR proteins assemble into a single asymmetric complex, with the two component rings exhibiting differential proteolytic functionalities and adaxial surfaces; functional consequences are discussed. To determine the consequences of the partial loss of Clp protease activity, the leaf proteomes of wild-type and a CLPP3 null mutant were compared using MS-based spectral counting. 2116 proteins and protein families were quantified and their differential expression in the mutant was tested for significance. This showed a general up-regulation of proteins involved in chloroplast proteome homeostasis and gene expression, but down-regulation of the photosynthetic machinery and specific responses of secondary metabolism. This demonstrates the essential contribution of ClpP3 in Clp core assembly and function, as well as the crucial role of the Clp core complex in chloroplast viability. Large-scale, label-free quantification was used to characterize large (>800 kDa) soluble, chloroplast-localized protein and nucleoprotein assemblies in Arabidopsis thaliana which were separated by size exclusion chromatography. Hierarchical clustering using MS-derived spectral counts for each chromatography fraction effectively grouped the identified proteins into functional complexes. This combined experimental and bioinformatics analyses resolved chloroplast chromatin, numerous novel proteins, as well as chloroplast ribosomes in different assembly and functional states, with ribosome assembly factors and proteins involved in cotranslational modifications, targeting and folding