REP27, a tetratricopeptide repeat nuclear-encoded and chloroplast-localized protein, functions in D1/32-kD reaction center protein turnover and photosystem II repair from photodamage

Abstract

The goal of this research is elucidation of the molecular mechanism for the unique photosystem II (PSII) damage and repair cycle in chloroplasts. A frequently occurring, irreversible photooxidative damage inhibits the PSII charge separation reaction and stops photosynthesis. The chloroplast PSII repair process rectifies this adverse effect by selectively removing and replacing the photoinactivated D1/32-kD reaction center protein (the chloroplast-encoded psbA gene product) from the massive (>1,000 kD) water-oxidizing and O2-evolving PSII holocomplex. DNA insertional mutagenesis in the model organism Chlamydomonas reinhardtii was applied for the isolation and characterization of rep27, a repair-aberrant mutant. Gene cloning and biochemical analyses in this mutant resulted in the identification of REP27, a nuclear gene encoding a putative chloroplast-targeted protein, which is specifically required for the completion of the D1 turnover process but is not essential for the de novo biogenesis and assembly of the PSII holocomplex in this model green alga. The REP27 protein contains two highly conserved tetratricopeptide repeats, postulated to facilitate the psbA mRNA cotranslational insertion of the nascent D1 protein in the existing PSII core template. Elucidation of the PSII repair mechanism may reveal the occurrence of hitherto unknown regulatory and catalytic reactions for the selective in situ replacement of specific proteins from within multiprotein complexes.

Figure 1.

Phenotypic characterization of C. reinhardtii wild-type (cw15) and rep27 mutant strains. A, Growth of cw15 wild type and lack of growth of the rep27 mutant on TBP minimal media at 50 μmol photons m⁻² s⁻¹ (50 μE). B, Growth curves of wild type and rep27 in TAP liquid media under approximately 10- or 50-μE conditions. The values shown are the average of the three independent experiments. C, Efficiency of PSII primary photochemistry (F_v/F_m) measured as a function of growth irradiance for wild type and rep27 mutant. D, Light-saturated rate of oxygen evolution for wild type and rep27 mutant as a function of growth irradiance. Cells were grown in TAP for 3 d prior to the measurement. Values shown are the average from the two independent experiments.

Figure 2.

A, Western-blot analysis of 50-μE grown C. reinhardtii wild type and rep27 total protein cell extract. The steady-state level of PSII subunits was determined from the intensity of the antibody cross-reaction in the western blots with specific polyclonal antibodies generated against D1, D2, CP47, and PsbO, respectively. B, Western-blot analysis of 50-μE grown wild type and rep27 total protein cell extract. The steady-state level of these thylakoid membrane proteins was determined from the intensity of the antibody cross-reaction in the western blots with specific polyclonal antibodies generated against PsaK, Cyt f, PetC, AtpA, and Hsp70B.

Figure 3.

A, Northern-blot analysis of total RNA from 50-μE grown wild-type and rep27 mutant strains separated onto 1.5% agarose gel. Following transfer to a nylon membrane, samples were treated with radiolabeled psbA and psbD cDNA. The C. reinhardtii rRNA loading control is shown at the bottom. B, Autoradiogram of [³⁵S]-radiolabeled proteins in C. reinhardtii. Chloroplast protein biosynthesis was conducted in vivo under 50 μE illumination for a period of 10 min (pulse) in the presence of [³⁵S]-Na₂SO₄. [³⁵S]-radiolabeled proteins were visualized upon exposure of the SDS-PAGE onto a radioactivity detector. The protein positions corresponding to D1, D2, Rubisco large subunit (LSU), and ATP synthase α-subunit (CF1) are denoted. Minor rep27-specific protein bands are denoted by asterisk, while black circles denote wild-type-specific bands.

Figure 4.

A, Southern-blot analysis of C. reinhardtii genomic DNA using the NdeI/NdeI 3′ end of the Arg-7 gene on NcoI or HpaI digested wild-type and rep27 genomic DNA. Black arrows indicate the position of the intrinsic inactivated ARG7 genes in the wild-type and rep27 DNA and white arrows show the position of a single additional band resulting from the insert DNA in the rep27 mutant. B, C. reinhardtii genomic DNA map, derived from the JGI Chlamydomonas Genome Project, showing the relative position of five ORFs (scaffold 8, contigs 25 and 26) and the insertion locus of pJD67. Restriction enzyme sites (NcoI and HpaI) used in the digestion of genomic DNA and the position of probes for Southern-blot analysis (NdeI/NdeI) are indicated on the pJD67 plasmid. The dotted rectangle in the 5′ end of the pJD67 shows the missing pBluescript portion, apparently deleted during the pJD67 insertion. Also indicated by opposite-facing arrows is the location of six primer sets (RT0–RT5). Four subclones (1A, 2A, 4A, and 8) isolated from BAC clone 10A5 were used for the rep27 complementation experiments. TAIL-PCR amplified fragment (TAIL) and the position of the TPB primer for the TAIL-PCR also shown.

Figure 5.

A, Growth of wild type, rep27 mutant, and BAC clone 10A5 complemented rep27 strains on TBP minimal media under 50-μE irradiance. BAC clone 10A5, which contains the 25-kb deleted genomic DNA fragment, successfully rescued the acetate-requiring mutant phenotype in rep27 (BAC-T). BAC-C was randomly selected as a negative control for BAC-T. B, PCR characterization of BAC-T and BAC-C genomic DNA. PCR amplification of BAC-T and BAC-C was performed with a variety of different marker primer sets (RT0–RT5), as indicated.

Figure 6.

Physiological and biochemical analyses of C. reinhardtii wild-type, rep27, and rep27-comp strains. A, Strains were grown on TAP under 10 μE and transferred to 1,000 μE for different periods of time. F_v/F_m was plotted as a function of incubation time in 1,000 μE. Values shown are the average of two independent experiments. B, The light-saturation curves of photosynthesis in wild-type, rep27, and rep27-comp strains grown in TAP under 10- or 50-μE conditions. Registration of the dark respiration in the cell suspension was followed by 5-min measurements of the rate of oxygen evolution at 500, 1,000, 1,500, 2,000, and 2,500 μE. The values shown are the average of two independent experiments. C, Western-blot analysis of total cell protein extracts from wild-type, rep27, and rep27-comp strains loaded on an equal Chl basis. Proteins were probed with specific anti-D1 polyclonal antibodies.

Figure 7.

Deduced amino acid sequence alignments (ClustalW analysis; Higgins et al., 1994) of REP27 and its homologs. A, Database searches revealed that the deduced amino acid sequence of rice (Os01g0358300), Arabidopsis (At1g02910), and Ostreococcus (CAL55849) gene products are orthologs to that of REP27. Asterisks mark absolutely conserved residues among the four protein sequences. High and low similarity residues are marked by double or single dots, respectively. Vertical arrow indicates the predicted cleavage site of the chloroplast transit peptide targeting sequence. Solid lines and dotted lines over the amino acid sequences indicate the TPR motifs and predicted transmembrane helices, respectively. B, Phylogenetic tree showing the relative evolutionary proximity of the REP27 ortholog (Os01g0358300, At1g02910, and CAL55849) and paralog proteins (Os04g0507100 [rice], At2g28740 [Arabidopsis], CAL58275 [Ostreococcus], and C_142189 [Chlamydomonas]) based on the amino acid sequence comparisons.

Figure 8.

A, Northern-blot analysis probed with C. reinhardtii REP27-specific DNA. Wild-type cells, grown under 50-μE conditions (L), were incubated under high light (1,000 μE, H) for 1 h. Agarose gel lanes were loaded with 4 or 8 μg of total RNA from the 50 μE-grown (L) and 1,000 μE-exposed (H) cells, transferred to nylon membrane, and probed with a radiolabeled full-size REP27 cDNA probe. A (bottom), rRNA loading control. B, Northern-blot analysis probed with C. reinhardtii ELIP-specific DNA. Wild-type and rep27 mutant grown under 50-μE conditions were incubated under high light (1,000 μE) for 1 h. Agarose gel lanes were loaded with 4 μg of total RNA from the 50 μE-grown (L) and 1,000 μE-exposed (H) cells, transferred to nylon membrane, and probed with a radiolabeled full-size ELIP cDNA probe. Ribosomal RNA loading controls are shown at the bottom.

Figure 9.

Schematic of the putative function of the REP27 protein in the D1 protein turnover and PSII repair process. REP27 is shown to play a role in the cotranslational insertion of a nascent D1 (pD1) in the D1-less PSII template during the PSII repair cycle. This step follows the PSII disassembly and specific degradation of photodamaged D1 in the thylakoid membrane.