TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii

Abstract

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.

Figure 1.

Antibody characterization and analysis of TEF30 oligomer formation, cellular abundance, and intracellular localization. A, Immunodetection of TEF30 in cw15-325 whole-cell proteins (WC) corresponding to 2 µg of chlorophyll compared with 0.25 ng of recombinant (Rec.) TEF30 protein. B, Subcellular localization of TEF30 by immunoblotting. Ten or 3 µg (depending on the antiserum used) of protein from whole cells, chloroplasts (cp), and mitochondria (mt) isolated from strain cw15-302 was separated by SDS-PAGE and immunodetected with antisera against TEF30, mitochondrial carbonic anhydrase (mtCA), and stromal HSP70B. C, Microscopy images taken from strain cw15-325. Shown from left to right are differential interference contrast images, 4′,6-diamino-phenylindole (DAPI) staining, immunofluorescence, merge of DAPI and immunofluorescence, and a schematic drawing that combines information from the images. Antisera used for immunofluorescence were against HSP70B and HSP70A as markers for stroma and cytosol, respectively, and against TEF30. C, Cytosol; f, flagella; P, pyrenoid. Immunofluorescence signal is shown in green. D, One to 8 ng of recombinant proteins TEF30 (purified from E. coli) and D2 (purchased from Agrisera) was separated by SDS-PAGE together with cw15-325 whole-cell proteins corresponding to 0.125 to 2 µg of chlorophyll and immunodetected with antibodies against TEF30 and D2. E, Recombinant TEF30 protein was incubated with EDC/N-hydroxysuccinimide, separated on an SDS-polyacrylamide gel, and immunodetected with antibodies against TEF30.

Figure 2.

Localization of TEF30 in thylakoids of C. reinhardtii wild-type and mutant cells. A, Salt treatments during cell fractionation. Whole cells (WC) of strain cw15-325 were resuspended in lysis buffer (LB) containing the indicated salts, subjected to freezing/thawing, and separated into soluble (S) and pellet (P) fractions. One sample resuspended in lysis buffer only was subjected to sonication instead of freezing/thawing. Proteins were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and against integral membrane protein cytochrome f (Cytf) and stromal CGE1 as controls. B, Trypsin treatment of thylakoid membranes. Isolated thylakoids from strain cw15-325 were incubated on ice with trypsin, separated by SDS-PAGE, and immunodetected with antibodies against TEF30 and the peripheral CF1β subunit of the ATPase complex, lumenal plastocyanin (PCY1), and integral membrane protein cytochrome f as controls. C, Cell fractionation of various photosynthesis mutants. Whole cells of the wild type (WT) and thylakoid membrane protein mutants were separated into soluble and pellet fractions via freezing/thawing. Proteins were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and against integral membrane protein cytochrome f or D1 and stromal CGE1 as controls.

Figure 3.

Copurification of TEF30 with PSII particles. A, Nickel-affinity purification of PSII particles. Detergent-solubilized thylakoid membranes from a C. reinhardtii strain harboring a hexa-His-tagged D2 protein (D2His) and from wild-type control strain cc124 (WT) were subjected to nickel-affinity purification. Thylakoid membrane input proteins corresponding to 1 µg of chlorophyll and 4% of the imidazole eluate were separated by SDS-PAGE and immunodetected with antibodies against TEF30 and proteins representative for the major thylakoid membrane complexes. B, Silver staining of proteins immunoprecipitated from hexa-His-tagged PSII particles with preimmune serum (Pre) and antibodies against TEF30. C, Immunodetection of proteins immunoprecipitated from PSII particles. Purified PSII particles equivalent to approximately 1 µg of chlorophyll (Input) and 20% of the immunoprecipitates obtained with preimmune serum and antibodies against TEF30 (IP) were separated by SDS-PAGE and immunodecorated with the antisera indicated.

Figure 4.

Comigration of TEF30 with PSII core subunits as revealed by Suc density gradient centrifugation and blue native (BN)-PAGE. A, Thylakoid membranes from strain cw15-325 (1 mg chlorophyll mL⁻¹) were solubilized with 1% β-dodecyl maltoside (β-DDM) and separated on a linear 0.2 to 0.5 m Suc gradient at 205,000g for 16 h. Proteins were collected in 25 fractions, separated by SDS-PAGE, and detected with Colloidal Blue staining (top gel) or immunologically with the antisera indicated (bottom gels). B, Analysis of thylakoid membrane protein complexes by Suc density gradient centrifugation as described in A on wild-type (WT) strain cc124, PSI mutant F23, and PSII mutant ΔpsbA/D. C, Analysis of thylakoid membrane protein complexes by BN-PAGE. Thylakoids from wild-type strain cc124, PSI mutant F23, and PSII mutant nac2 were solubilized with 1% β-DDM, and proteins corresponding to 5 µg of chlorophyll were separated on a 5% to 15% polyacrylamide BN gradient gel, followed by Coomassie Blue staining (left gel) and immunodetection with antibodies against TEF30 (right gel). Protein bands were assigned to LHCII monomers (#1), LHCII trimers (#2), PSII monomers (#3), PSII dimers (#4), PSI supercomplexes (#5), and PSII supercomplexes (#6) according to Järvi et al. (2011).

Figure 5.

PSII activity and levels of PSII core subunits are more sensitive to HL intensities or low temperatures in TEF30-amiRNA cells than in control cells. A, cw15-325 control (Con) and TEF30-amiRNA strains were photoinhibited (PI) for 60 min at 1,800 μmol photons m^–2 s^–1, then allowed to recover at 30 μmol photons m^–2 s^–1. PSII fluorescence was measured by pulse amplitude-modulated fluorometry. Values represent means from three biological replicates, and error bars indicate sd. B, Control and TEF30-amiRNA #1.48 cells in the cw15-325 strain background were exposed to high PFDs (HL) of approximately 800 µmol photons m⁻² s⁻¹ for 6 h. Whole-cell proteins corresponding to 0.2, 1, or 2 µg of chlorophyll (depending on the antiserum used) were separated on 10% or 14% SDS-polyacrylamide gels and analyzed by immunoblotting. C, Control and TEF30-amiRNA #2.48 cells in the cw15-325 strain background were grown at a PFD of approximately 30 μmol photons m^–2 s^–1 and then shifted from a growth temperature of 25°C to 10°C for 18 h. Samples were analyzed as in B.

Figure 6.

Comparison of LHCSR3 expression and oxygen evolution in control and TEF30-amiRNA cells. A, Time course of LHCSR3 accumulation at 600 μmol photons m^–2 s^–1 (HL) in cw15-325 control (Con) and TEF30-amiRNA cells grown in Tris-acetate-phosphate (TAP)-NH₄, TAP-NO₃, or Tris-minimal-phosphate (TMP)-NH₄. Whole-cell proteins were separated by SDS-PAGE and immunodetected with antibodies against LHCSR3 and against CF1β as loading control. B, Oxygen evolution from thylakoid membranes isolated from cw15-325 control and TEF30-amiRNA cells grown in TAP-NH₄ medium at LL of approximately 30 µmol photons m⁻² s⁻¹ or HL of approximately 800 µmol photons m⁻² s⁻¹ for 4 h. Values represent means from experiments on two independent control and TEF30-amiRNA lines, and error bars indicate sd. (Note that the same thylakoid preparation was used for all PFDs applied, thus explaining the decline in oxygen evolution at 1,600 µmol photons m⁻² s⁻¹.)

Figure 7.

PSII de novo synthesis is not impaired in TEF30-amiRNA strains. A, TEF30-amiRNA and control (Con) cells in the cw15-325 background were grown in TAP-NH₄ at approximately 30 µmol photons m⁻² s⁻¹ to the exponential phase. Cells were then harvested, washed, and resuspended in TAP medium lacking sulfur (–S). After 48 h under –S conditions, cells were shifted back to regular TAP medium (+S) for another 24 h. Whole-cell proteins from samples taken during this time course were separated by SDS-PAGE, and PSII polypeptide abundance was measured via immunoblotting using the antibodies indicated and CF1β as a loading control. B, D1 signal intensities from A were quantified using the FUSIONCapt Advance program. Signals were normalized to the initial D1 levels in each strain. Error bars represent sd; n = 2. C, Sulfur depletion was done as in A, but time in –S and +S conditions was reduced to 24 and 6 h, respectively. Thylakoids were solubilized with 1% α-dodecyl maltoside (α-DDM), and proteins corresponding to 5 µg of chlorophyll were separated on a 5% to 15% polyacrylamide BN gradient gel, followed by Coomassie Blue staining (top gel) and immunodetection with antibodies against D1 and CP43 (bottom gels). Protein bands were assigned as in Figure 4C; the asterisk designates free CP43. D, D1 signal intensities from protein bands #6 (PSII supercomplexes) and #4 (PSII dimers) from C were quantified using the FUSIONCapt Advance program. Signals were normalized to the initial D1 levels in each strain. Error bars represent sd; n = 4.

Figure 8.

PSII stability is not impaired in TEF30-amiRNA strains. A, TEF30-amiRNA and control (Con) cells in the cw15-325 background were grown at approximately 30 µmol photons m⁻² s⁻¹ in TAP medium in the presence or absence of 100 µg mL⁻¹ CAP for 12 h. Proteins from whole cells harvested during the time course corresponding to 0.5 or 1 µg of chlorophyll (depending on the antiserum used) were analyzed by immunoblotting. D1 levels were quantified with the FUSIONCapt Advance program. D1 levels were normalized to the values before the addition of CAP (at 0 h), and the ratio between D1 levels from TEF30-amiRNA strains and controls was calculated. Values represent means from four independent replicates, and error bars indicate sd. B, TEF30-amiRNA and control cells in the cw15-325 background were grown in TMP minimal medium at 200 μmol photons m^–2 s^–1 in the presence of 100 µg mL⁻¹ CAP for 12 h. Analyses were done as in A, with two biological replicates. C, TEF30-amiRNA and control cells in the cw15-325 background were grown in TAP medium in the dark in the presence of 100 µg mL⁻¹ CAP for 72 h. Analyses were done as in A, with two biological replicates.

Figure 9.

Not PSII sensitivity to HL, but PSII repair, is impaired in TEF30-amiRNA strains. A, Cells of cw15-325 control (Con) and TEF30-amiRNA strains were photoinhibited (PI) for 60 min at 1,800 μmol photons m^–2 s^–1 in the presence of the chloroplast translation inhibitors CAP and lincomycin and allowed to recover at 30 μmol photons m^–2 s^–1 (LL) without inhibitors. PSII fluorescence was measured by pulse amplitude-modulated fluorometry. Values represent means from two biological replicates, and error bars indicate sd. B, Immunoblot analysis of whole-cell proteins collected from the experiments described in A. Results from a typical experiment are shown at the top, and means from quantified D1 signals (normalized relative to the initial D1 signal) are shown at the bottom. Values represent means from five biological replicates, and error bars indicate sd.

Figure 10.

PSII supercomplex accumulation is impaired in TEF30-amiRNA strains exposed to HL. cw15-325 control (Con) and TEF30-amiRNA cells were grown at 30 µmol photons m⁻² s⁻¹ (LL) and exposed to 800 µmol photons m⁻² s⁻¹ (HL) for 4 h. Thylakoid membranes were isolated and solubilized with 1% α-DDM, and protein complexes were separated electrophoretically on a 7.5% native Deriphat-polyacrylamide gel. The gel was then photographed (left) and processed for immunoblotting using antibodies against TEF30 and the D1 protein (right). Bands were assigned to free pigments (#0), antenna monomers (#1), LHCII trimers (#2), PSI/II cores (#3), and PSI/II supercomplexes (#4) according to Formighieri et al. (2012).

Figure 11.

Cells of TEF30-amiRNA strains contain fewer thylakoid membranes per stack. A, Electron microscopy images of cw15-325 cells characteristic for the categories normally stacked, reduced stacking, and swollen. Overview images are shown on the left, and enlarged images of the region demarcated by the black box are shown on the right. N, Nucleus; P, pyrenoid; S, starch. Bars in overview images = 1 µm, and those in enlarged images = 0.2 µm. B, cw15-325 control (Con) and TEF30-amiRNA cells were grown in TAP medium at approximately 30 µmol photons m⁻² s⁻¹ (LL) and exposed to approximately 800 µmol photons m⁻² s⁻¹ (HL) for 4 h. Fifty electron micrographs each for two independent control strains and three independent TEF30-amiRNA strains were taken for each condition, and thylakoid phenotypes were assessed according to the categories shown in A. Values are given in percent and represent means from the two control and three TEF30-amiRNA strains. Error bars indicate sd. Significance was tested using Student’s t test (*, P < 0.05 and **, P < 0.01).

Figure 12.

Model for TEF30 function during PSII repair. When PSII is damaged (e.g. at HL intensities), the PSII complex enters the repair cycle. For this, PSII supercomplexes are disassembled and PSII monomers migrate from stacked into stroma-exposed membrane regions. After PSII monomers are partially disassembled to generate CP43-less forms, damaged D1 is degraded by FtsH and Deg proteases and a newly synthesized D1 copy is inserted into PSII monomers. At this step, TEF30 may facilitate incorporation of the new D1 protein and/or reassembly of CP43, bind to repaired PSII monomers to protect them from undergoing repeated repair cycles, or facilitate the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly. These functions are not mutually exclusive.