Article Figures & Data
Figures
- Fig. 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.
- Fig. 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−1for induction of LHCII phosphorylation (C).
- Fig. 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.
- Fig. 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 b 559 (Cytb 559), 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.
- Fig. 5.
Temperature dependence of the phosphatase activity of the isolated membrane enzyme and the enzyme bound to thylakoid membranes. Phosphatase assays were performed with32P-labeled phosphopeptides as a substrate. The32P-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.
- Fig. 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).
Tables
- Table I.
Temperature dependence of the dephosphorylation rates for the major phosphoproteins in isolated thylakoids
Temperature t1/2 22°C 27°C 35°C 42°C min Phosphoprotein CP43 22 ± 8 33 ± 5 9 ± 4 2 ± 0.6 D1/D2 49 ± 12 49 ± 7 8 ± 4 4 ± 2 9-kD 52 ± 26 55 ± 10 9 ± 6 19 ± 10 LHCII 35 ± 10 37 ± 7 12 ± 10 18 ± 7 29-kD 43 ± 9 35 ± 8 14 ± 4 13 ± 3 The data are presented as the half-times (minutes). The half-times were calculated from the first-order rate fitting of the dephosphorylation versus time curves obtained from four experiments at each temperature.
