Mass Spectrometric Resolution of Reversible Protein Phosphorylation in Photosynthetic Membranes ofArabidopsis thaliana

Alexander V Vener,Amy Harms,Michael R Sussman,Richard D Vierstra

doi:10.1074/jbc.m009394200

Alexander V Vener, Amy Harms + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m009394200

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Abstract

The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.

Highlights

The reversible phosphorylation of specific proteins participates in the regulation of virtually all aspects of cell physiology and development
From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry
These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3

Summary

Mass Spectrometry of Thylakoid Protein Phosphorylation

Versible phosphorylation is thought to play critical roles in (i) the redistribution of excitation energy between PSI and II via modification of LHCII [11,12,13], and (ii) the maintenance of the PSII by controlling the turnover of its reaction center polypeptides (14 –18). Studies in spinach and pea using 32P labeling and phosphoamino acid antibodies showed that a number of proteins are phosphorylated, including threonine residues at or near the N termini of LHCII [19] and PSII polypeptides, the D1 and D2 reaction center proteins, chlorophyll-binding protein CP43 [20], and peripheral polypeptide PsbH [21]. In vitro studies using 32P labeling have suggested that a number of thylakoid proteins are extensively phosphorylated in the light [22] by a kinase controlled by the photosynthetic electron transport chain [12, 23, 24], and dephosphorylated in the dark by phosphatase(s) that are not light sensitive [12, 25]. The MS techniques described here should be applicable for many analyses involving complex mixtures of phosphoproteins

EXPERIMENTAL PROCEDURES

Enriched by Peptide mass IMAC with Parent protein

Phosphorylation levelb

DISCUSSION