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, 123 (4), 1525-36

Dephosphorylation of Photosystem II Reaction Center Proteins in Plant Photosynthetic Membranes as an Immediate Response to Abrupt Elevation of Temperature

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Dephosphorylation of Photosystem II Reaction Center Proteins in Plant Photosynthetic Membranes as an Immediate Response to Abrupt Elevation of Temperature

A Rokka et al. Plant Physiol.

Abstract

Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22 degrees C to 42 degrees C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll a binding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2- to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSII-specific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidyl-prolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts.

Figures

Figure 1
Figure 1
Dephosphorylation of thylakoid proteins in vitro at 22°C and 42°C. Spinach thylakoid membranes were isolated and phosphorylated in the presence of [γ-32P]ATP under a photon flux density (PFD) 300 μmol photons m−2 s−1. The autoradiograms show dephosphorylation of thylakoid phosphoproteins by endogenous phosphatases in darkness either at 22°C (A) or 42°C (B). Positions of the major thylakoid phosphoproteins are indicated.
Figure 2
Figure 2
Dephosphorylation of thylakoid proteins in vivo at 22°C and 42°C. Spinach leaf discs were illuminated 60 min at 22°C and then transferred to darkness and incubated at 22°C or 42°C. Dephosphorylation was terminated at the indicated time points by freezing the leaf discs in liquid nitrogen. Thylakoid membranes were isolated and the extent of protein phosphorylation was determined using a P-Thr antibody (A and C) or a D1-specific antibody (B). In the latter case the upper band of the D1 doublet represents the phosphorylated form of the protein, indicated by PO3-D1. Before conducting the dephosphorylation experiments different light intensities were used for induction of higher in vivo phosphorylation levels of either PSII core proteins or LHCII. The leaf discs were illuminated under a PFD 1,000 μmol photons m−2 s−1 for more effective phosphorylation of PSII proteins (A, B) or under a PFD 80 μmol photons m−2 s−1 for induction of LHCII phosphorylation (C).
Figure 3
Figure 3
Dephosphorylation of thylakoid proteins in NaBr-washed thylakoids. Radioactively labeled thylakoid membranes were washed with 2 m NaBr to remove extrinsic protein phosphatases. Subsequently the thylakoids were incubated in darkness either at 22°C (A) or 42°C (B) to follow protein dephosphorylation as presented on autoradiograms.
Figure 4
Figure 4
Lateral migration of the PSII induced by high temperature. Isolated thylakoid membranes (T) incubated for 5 min at 22°C or 42°C were subfractionated into grana (G), grana margins (Gm), and stroma-exposed thylakoids (S) using digitonin and differential centrifugation. Marker PSII proteins, CP43, the D1 protein, and cytochrome b559 (Cyt b559), were detected using specific corresponding antibodies. An antibody against ATP synthase subunit CFo was used as a control for proteins with permanent location in stroma-exposed thylakoid membranes and grana margins.
Figure 5
Figure 5
Temperature dependence of the phosphatase activity of the isolated membrane enzyme and the enzyme bound to thylakoid membranes. Phosphatase assays were performed with 32P-labeled phosphopeptides as a substrate. The 32P-labeled phosphopeptides were obtained from radioactively labeled thylakoid membranes by trypsin treatment. The phosphatase activity of isolated phosphatase (A) and of intact thylakoids (B) was measured at 22°C, 27°C, 35°C, or 42°C. The initial phosphopeptide concentrations were 10 μm, based on the 32P content. C, Comparison of the increase in dephosphorylation rate constants at different temperatures for the isolated phosphatase and the membrane-bound enzyme.
Figure 6
Figure 6
Release of TLP40 from thylakoid membranes as a result of a heat treatment. Isolated thylakoid membranes were incubated 5 min at 22°C or 42°C and then frozen in liquid nitrogen. Subsequently the membranes were disrupted with DM, and the membrane fraction and lumenal fraction, containing the released proteins, were separated by centrifugation. The proportions of membrane-bound and released TLP40 were determined with a specific antibody. As a control the content of the Rieske iron-sulfur protein was determined in the same fractions. The Rieske protein is located in thylakoid lumen and bound to the membrane via a single transmembrane anchoring span (Karnauchov et al., 1997).

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