Abstract
We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for in vitro phosphorylation experiments and specifically assessed the peptide substrate spectrum of chloroplast casein kinase II (pCKII). To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.
Highlights
Chloroplasts have to respond quickly to changes in light quality and quantity in order to balance the excitation pressure between the two photosystems
We extracted information on Arabidopsis thaliana chloroplast phosphoproteins from different published phosphoproteomics experiments at the status of January 2012, most of them being represented in the PhosPhAT 3.0 database
In order to ensure accessibility of phosphorylation sites, we centered the peptides on the phosphorylated amino acid, i.e. we added 7 amino acids upstream and downstream to the phosphorylation site as determined by mass spectrometry
Summary
Chloroplasts have to respond quickly to changes in light quality and quantity in order to balance the excitation pressure between the two photosystems. Important aspects of this fast responding system are controlled by posttranslational modifications of resident proteins, e.g. phosphorylation (reviewed in [1,2,3]). Several chloroplast protein kinases are known that catalyze light-triggered acclimation responses, but their complete set of targets and their cross talk in a phosphorylation network are only partially understood. In addition to its role in controlling state-transitions, STN7 is involved in the regulation of long-term acclimation processes that entail changes in gene expression of both nuclear and chloroplast encoded genes [4]. It appears likely that a direct control of plastid gene expression is exerted by other kinases such as the chloroplast sensor kinase CSK or casein kinase II [2]
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