Mutation of residue threonine-2 of the D2 polypeptide and its effect on photosystem II function in Chlamydomonas reinhardtii

Abstract

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Figure 1

Restriction map of a 7.5-kb HindIII fragment from the chloroplast genome of C. reinhardtii showing the mutations made to the psbD gene. The positions and orientations of psbD, psaA-2, and the spectinomycin-resistance cassette (spec^R) are indicated. Restriction sites are HindIII (H), EcoRI (R), NsiI (N), HpaI (Hp), and MunI (M). The MunI site eliminated along with the creation of the Thr-2 mutations is shown in bold.

Figure 2

Southern-blot analysis of the wild type (lanes 1 and 5), Nsi16 (lanes 2 and 6), and mutants Thr2Ser (lanes 3 and 7) and Thr2Ala (lanes 4 and 8). ctDNA was digested with HindIII (lanes 1–4) and MunI (lanes 5–8), and hybridized to a 0.6-kb psbD probe.

Figure 3

Relaxation of F_v in the Thr-2 mutants. A, Relaxation of the F_v after each of a series (1.67 Hz) of five saturating 2-μs flashes in whole cells of the C. reinhardtii mutants Thr2Ala and Thr2Ser. Shown is the 5-ms range after each flash starting at 50 μs. The 50-μs values of F_v are normalized to F_o [(F − F_o)/F_o]. B, Relaxation of F_v resulting from charge recombination between Q_A⁻ and the PSII donor side after a single, saturating 2-μs flash excitation of whole cells of C. reinhardtii. Wild type (•) is compared with transformant Nsi16 (×) and with the mutants Thr2Ala (⋄) and Thr2Ser (□).

Figure 3

Relaxation of F_v in the Thr-2 mutants. A, Relaxation of the F_v after each of a series (1.67 Hz) of five saturating 2-μs flashes in whole cells of the C. reinhardtii mutants Thr2Ala and Thr2Ser. Shown is the 5-ms range after each flash starting at 50 μs. The 50-μs values of F_v are normalized to F_o [(F − F_o)/F_o]. B, Relaxation of F_v resulting from charge recombination between Q_A⁻ and the PSII donor side after a single, saturating 2-μs flash excitation of whole cells of C. reinhardtii. Wild type (•) is compared with transformant Nsi16 (×) and with the mutants Thr2Ala (⋄) and Thr2Ser (□).

Figure 4

Photoinhibition measurements. Effect of high-light treatment (1000 μmol m⁻² s⁻¹) on the light-saturated rate of oxygen evolution from whole cells of the wild type, Nsi16, and the mutants Thr2Ala and Thr2Ser in the presence of 200 μg/mL CAP. Cells were suspended in HSM at 25°C to a chlorophyll concentration of 25 μg/mL. Oxygen evolution was measured using 1 mm DCBQ and 1 mm potassium ferricyanide. The 100% rates of oxygen evolution were approximately 251 μmol O₂ mg⁻¹ chlorophyll h⁻¹ for the wild type (•), 356 μmol O₂ mg⁻¹ chlorophyll h⁻¹ for Nsi16 (×), 222 μmol O₂ mg⁻¹ chlorophyll h⁻¹ for Thr2Ala (○), and 333 μmol O₂ mg⁻¹ chlorophyll h⁻¹ for Thr2Ser (□).

Figure 5

In vitro labeling of wild-type thylakoid membranes with [γ-³²P]ATP. A, Autoradiogram of a 12%, 6 m urea-SDS gel. B and C, Immunoblotting with α-LHCII (B) and α-D2 (C) antibodies. Lanes 1, Cells grown in the light and thylakoids labeled in the light; lanes 2, cells grown in the light and thylakoids labeled in the dark; lanes 3, cells grown in the dark and thylakoids labeled in the light; and lanes 4, cells grown in the dark and thylakoids labeled in the dark. Arrows indicate positions of main LHCII bands picked up by the α-LHCII antibody. The triangle represents the position of CP43. Dots indicate unidentified phosphoproteins. Low-molecular-mass polypeptides have been run off the bottom of the gel. Numbers next to the α-D2 blot represent the bands of the molecular-mass marker in kilodaltons (prestained LMW, Bio-Rad).

Figure 6

In vitro labeling of wild-type (lanes 1–4) and Thr2Ala (lanes 5–8) thylakoid membranes with [γ-³²P]ATP. A, Autoradiogram of a 12%, 6 m urea-SDS gel. B, Immunoblotting with α-D2 antibody. Lanes 1 and 5, Cells grown in the light and thylakoids labeled in the light; lanes 2 and 6, cells grown in the light and thylakoids labeled in the dark; lanes 3 and 7, cells grown in the dark and thylakoids labeled in the light; lanes 4 and 8, cells grown in the dark and thylakoids labeled in the dark. Arrows indicate positions of main LHCII bands picked up by the α-LHCII antibody as shown in Figure 5. The triangle represents the position of CP43. Dots indicate unidentified phosphoproteins. Numbers next to the α-D2 blot represent the bands of the molecular-mass marker in kilodaltons (prestained LMW, Bio-Rad).

Figure 7

In vivo labeling of C. reinhardtii CC2655 cells with ³²Pi using 10 to 17% Coomassie blue-stained, 6 m urea-SDS gel (A) and autoradiography (B). Lane M, Molecular-mass marker in kilodaltons (prestained LMW, Bio-Rad); lanes 1, thylakoid membranes; lanes 2, isolated PSII RCs; lane 3, PSII RCs from pea. The autoradiogram was obtained by exposing the gel to a Phosphor Imager screen (Molecular Dynamics) for 7 d. The amount of chlorophyll was 5 μg for the CC2655 thylakoids, 1 μg for the CC2655 PSII RCs, and 0.2 μg for pea PSII RCs.

Figure 8

Pulse-chase labeling of whole cells with [¹⁴C]acetate. Autoradiogram (A) and α-D2 immunoblot of a 14%, 8 m urea-SDS gel (B) of thylakoid membranes isolated from wild-type, Nsi16, Thr2Ala, and Thr2Ser cells pulse labeled for 5 min with [¹⁴C]acetate at an incident light intensity of 100 μmol m⁻² s⁻¹ and then chased for 45 and 90 min in the absence of label. Both pulse and chase were carried out in the presence of 10 μg/mL cycloheximide.