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

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.The reversible phosphorylation of specific proteins participates in the regulation of virtually all aspects of cell physiology and development. The extent of its importance is illustrated by the hundreds of conventional protein kinases and phosphatases detected in various eukaryotic genomes (1-3). Whereas, serine, threonine, and tyrosine residues are the typical targets of these kinases, phosphorylation of at least six other amino acids is feasible, potentially expanding even further the dimensions of this post-translational modification (reviewed in Ref. 4). Despite the importance of this pool of phosphorylated proteins, our understanding of its depth and breadth remains sketchy. One barrier has been the lack of methods to define en masse the "phosphorylome," i.e. the subset of proteins in the proteome that become modified in vivo by phosphorylation. Precise characterization of the phosphorylome will be essential to fully understand how proteins are activated or inhibited, encouraged to interact with other components in the cell, and selected for rapid degradation. Certainly, the dynamic and transient nature of many protein phosphorylation reactions underscores the difficulties of resolving the complete phosphorylome for a given organism. Nevertheless, the identification of even just the principal cellular phosphoproteins under dis...

Structural and Functional Assays of AtTLP18.3 Identify Its Novel Acid Phosphatase Activity in Thylakoid Lumen

“…AtTLP18.3 showed phosphatase activity with two artificial substrates, pNPP and DiFMUP. To explore the possible cellular substrate activity, we confirmed the acid phosphatase activities with pSer and several designed phosphorylated oligopeptides from PSII (Vener et al, 2001). Theoretically, because the phosphorylated oligopeptides are located at the N terminus of PSII in the chloroplast stroma, their dephosphorylation by AtTLP18.3 should not occur in the thylakoid lumen.…”

“…We tested the phosphatase activity of AtTLP18.3 against five synthetic phosphorylated oligopeptides: Ac-(pT)AILER and Ac-(pT)IALGK are the phosphorylation sites of the major phosphopeptides of D1 and D2 proteins from Arabidopsis thylakoids (Vener et al, 2001); RRA(pT)VA and KR(pT)IRR are good substrates of the Ser/Thr phosphatases (Donella-Deana et al, 1991); and Ac-RK(pS)AGKPKN is the phosphorylation Figure 3. Overall structure and topology diagram of AtTLP18.3.…”

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

“…A newly synthesized D1 protein is then inserted into the PSII complex, which moves back to the grana region. This repair cycle requires a series of phosphorylation and dephosphorylation events at the level of the PSII core proteins D1, D2, CP43 and PsbH [7,8].…”

A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes

“…Two other subunits of PSII, the chlorophyll a-binding protein CP43 and the 9-kDa PsbH gene product, are also phosphorylated in thylakoid membranes (20, 21). All of the thylakoid phosphoproteins identified so far are hydrophobic integral membrane proteins phosphorylated at threonine residues at or near their N termini (12,21,22).One of the phosphopeptides in the thylakoid membrane, the so-called 12-kDa phosphoprotein (23, 24), has, however, resisted identification with respect to its sequence, corresponding gene, and functional significance. An earlier N-terminal sequencing of this protein from spinach revealed a unique stretch of amino acids that did not match any protein in the databases (24).…”