Cross-talk between calcium signalling and protein phosphorylation at the thylakoid

Abstract

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.

Fig. 1. Calcium-dependent phosphorylation of pea thylakoid proteins.

(A) The left panel displays a Pro-Q Diamond phosphoprotein gel stain that reveals three differentially phosphorylated protein bands (indicated with an asterisk) upon the addition of 1 mM Ca²⁺, compared with 1 mM EGTA. The right panel is the protein loading control (CBB=Coomassie Brilliant Blue). (B) 2D protein separation according to isoelectric point (pI) and size (kDa) of the same Ca²⁺-dependent phosphorylation assay followed by a Pro-Q Diamond phosphoprotein gel stain (experiment 1). Two protein spots were identified as the FtsH protease, Variegated 1 (VAR1) and ‘Calcium sensing’ protein (CAS).

Fig. 2. Calcium-dependent phosphorylation of Arabidopsis thylakoid proteins visualized by Pro-Q Diamond phosphoprotein gel stain (A) and phospho-threonine specific antibody (B) after 2D-gel separation according to isoelectric point (pI) and size (kDa).

(A) Protein spots identified in experiment 4 (250 μM Ca²⁺/EGTA) are the FtsH protease Variegated 2 (VAR2), ‘Calcium sensing’ protein (CAS), Photosystem I subunit P (PsaP), Photosystem I subunit H-2 (PsaH-2), and Photosystem I subunit N (PsaN; three times identified). (B) Protein spots identified in experiment 3 (250 μM Ca²⁺/EGTA) are the ATPase subunit F (ATPF), the Photosystem II subunits Q1 and Q2 (PsbQ1-2), PsaH-2, and PsaN. Stronger phosphorylation of spots were also observed in the EGTA samples, as it is the case in (A) for the ATPase C1 subunit (ATPC1) and in (B) for the MAR binding filament-like protein 1 (MFP1).

Fig. 3. Calcium-dependent phosphorylation assays on recombinant PsaN, CAS, VAR1, VAR2, and GST protein.

The two middle lanes of the left panel display the autoradiograph of ³²P-labelled proteins of a Ca²⁺-dependent phosphorylation assay of recombinant protein (prot) by an Arabidopsis chloroplast protein extract (chloro). The two left lanes are a control of protein only, whereas the two right lanes are a control for the background phosphorylation in the chloroplast protein extract. Note that GST is not being phosphorylated, instead a strong phosphorylation is visible in the EGTA sample of the chloroplast protein extract. The right panel is the protein loading control (CBB=Coomassie Brilliant Blue).