Probing the oligomeric state of the chloroplast proteome in Arabidopsis thaliana by mass spectrometry: megadalton-sized complexes and the Clp protease machinery

摘要

Size fractionation of the soluble chloroplast proteome followed by MS analysis catalogued protein assemblies between 0.8 and approx5 MDa (“HM” range). Proteins were grouped by hierarchical clustering based on their abundance distribution (measured from spectral counts) across the SEC fractions. The HM fractions were dominated in biomass by 30S-50S-70S ribosome particles and associated factors. Furthermore, the plastid chromosome with interacting proteins (nucleoid) was observed. Additional proteins that comprise or associate with these assemblies were also identified extending the chloroplast proteome coverage. These include proteins involved in ribosome biogenesis, translation as well as co-translational protein modification, folding and targeting. The low-abundant ribosome biogenesis factors that were identified include ribosome associated (RA)-GTPases similar to prokaryotic factors and plant-specific ribosome maturation proteins. Targeted genetic and biochemical analyses will uncover their roles in ribosome assembly. From MS-based absolute quantification of the Clp subunits using the QConCAT method, the stoichiometry of the Clp core and its sub-complexes was determined. The ClpP1/R ring is a distinct subcomplex and is composed of 3:1:1:1:1 (P1:R1:R2:R3:R4).