Mechanism of REP27 protein action in the D1 protein turnover and photosystem II repair from photodamage

Abstract

The function of the REP27 protein (GenBank accession no. EF127650) in the photosystem II (PSII) repair process was elucidated. REP27 is a nucleus-encoded and chloroplast-targeted protein containing two tetratricopeptide repeat (TPR) motifs, two putative transmembrane domains, and an extended carboxyl (C)-terminal region. Cell fractionation and western-blot analysis localized the REP27 protein in the Chlamydomonas reinhardtii chloroplast thylakoids. A folding model for REP27 suggested chloroplast stroma localization for amino- and C-terminal regions as well as the two TPRs. A REP27 gene knockout strain of Chlamydomonas, termed the rep27 mutant, was employed for complementation studies. The rep27 mutant was aberrant in the PSII-repair process and had substantially lower than wild-type levels of D1 protein. Truncated REP27 cDNA constructs were made for complementation of rep27, whereby TPR1, TPR2, TPR1+TPR2, or the C-terminal domains were deleted. rep27-complemented strains minus the TPR motifs showed elevated levels of D1 in thylakoids, comparable to those in the wild type, but the PSII photochemical efficiency of these strains was not restored, suggesting that the functionality of the PSII reaction center could not be recovered in the absence of the TPR motifs. It is suggested that TPR motifs play a role in the functional activation of the newly integrated D1 protein in the PSII reaction center. rep27-complemented strains missing the C-terminal domain showed low levels of D1 protein in thylakoids as well as low PSII photochemical efficiency, comparable to those in the rep27 mutant. Therefore, the C-terminal domain is needed for a de novo biosynthesis and/or assembly of D1 in the photodamaged PSII template. We conclude that REP27 plays a dual role in the regulation of D1 protein turnover by facilitating cotranslational biosynthesis insertion (C-terminal domain) and activation (TPR motifs) of the nascent D1 during the PSII repair process.

Figure 1.

Solubilized thylakoid membranes of Chlamydomonas wild type (CC425) were subjected to PAGE analyses. A (top lane), BN-PAGE of thylakoid membrane proteins stained with Coomassie Brilliant Blue. A (bottom lane), green native gel of the same sample. B, Two-dimensional gel electrophoresis of the wild type, where BN-PAGE was used in the first dimension (Dim.) and 12% acrylamide SDS-PAGE was used in the second dimension. The two-dimensional gel was visualized by silver staining. mA, Mitochondrial H⁺-ATP synthase; PII1 to PII3, PSII reaction center subunits (D1, D2, CP43, and CP47 proteins); PI, PSI complex (PsaA, PsaB, and PsaF) and LHCI protein; cA, chloroplast ATP synthase (CF₀F₁); Cb, cytochrome b₆f; LT, LHCII trimer; LM, LHCII monomer.

Figure 2.

Comparison of silver-stained two-dimensional gels between the wild type (CC425) and the rep27 mutant. The dashed lines demarcate the expected locations of the PSII core complex (CC425) and the LHCII (rep27). For more details, see legend to Figure 1.

Figure 3.

Western-blot analysis of Chlamydomonas wild-type (WT) and rep27 thylakoid membrane proteins. Cells were grown under continuous irradiance of 100 μmol m⁻² s⁻¹ incident intensity. Steady-state levels of PSII subunits were determined from the intensity of the antibody cross-reaction on western blots with specific polyclonal antibodies generated against D1, D2, CP47, CP43, REP27, and the ATP synthase (ATPsynt). Note the lower D1 and CP43 levels in rep27 and the absence of REP27 in the mutant.

Figure 4.

A, Western-blot analysis of total proteins from Chlamydomonas wild type (CC425), rep27, rep27-comp, D1-less (ΔpsbA), and D2-less (ΔpsbD) mutant strains probed with specific polyclonal antibodies raised against the REP27 protein. B, SDS-PAGE of proteins (2 μg of chlorophyll loaded) separated onto 12% acrylamide and visualized with Coomassie Brilliant Blue. The slightly slower electrophoretic mobility of the REP27 protein in the complemented strain (rep27-comp) is due to the presence of the 2×MYC tag, introduced into the plasmid construct used for complementation.

Figure 5.

Western-blot analysis of total proteins from Chlamydomonas wild-type (CC425), rep27, and rep27-comp strains. Cells were fractionated into soluble (S) and total membrane (TM) portions. Total extract (TE) was obtained upon breaking the cells with acid-washed glass beads. The total membrane fraction was obtained by centrifugation at 20,000g for 60 min. Lanes were loaded with 2 μg of chlorophyll. The supernatant was used as the soluble fraction. Relative protein content was estimated from the intensity of the antibody cross-reaction on the western blots. Specific polyclonal antibodies were used, generated against REP27, RbcL, or D1.

Figure 6.

Western-blot analysis of proteins from thylakoid membrane fractions isolated upon sonication and differential centrifugation at 10,000g (10k), 40,000g (40k), and 140,000g (140k). Lanes were loaded with 2 μg of chlorophyll (Chl). Proteins were probed with specific polyclonal antibodies generated against D1 (A) and REP27 (B). C shows a Coomassie Brilliant Blue-stained SDS-PAGE protein profile of the different thylakoid membrane fractions.

Figure 7.

Folding model of the REP27 protein in the chloroplast thylakoid membrane. This protein contains 449 amino acids from the N to the C terminus. The number of positively charged amino acids is indicated for each domain of the protein. Two transmembrane helices (TMH1 and TMH2) are shown spanning the thylakoid membrane such that N and C termini are exposed in the chloroplast stroma phase. The model further shows two TPR motifs (TPR1 and TPR2) occurring near the N terminus and the transit peptide that is cleaved upon chloroplast import. REP27 displays a rather extensive C-terminal portion, which is exposed in the chloroplast stroma. [See online article for color version of this figure.]

Figure 8.

REP27 cDNA constructs used for complementation of the rep27 mutant. On the left, the name of each construct is given. In the middle, a map of the structural organization of the respective construct is shown. On the right, transformant strains obtained from the transformation of rep27 mutant by these cDNA constructs are shown.

Figure 9.

Photoautotrophic growth of Chlamydomonas wild type, rep27 mutant, and rep27 transformants grown on HSM and TBP media. A, CC125 wild type; B, rep27 mutant; C, rep27-comp; D, rep27-ΔT1; E, rep27-ΔT2; F, rep27-ΔT1+2; G, rep27-ΔCt. [See online article for color version of this figure.]

Figure 10.

Growth (left) and efficiency of PSII primary photochemistry (F_v/F_m; right) of CC125 wild type, rep27 mutant, and rep27 transformant strains grown on TAP medium. F_v/F_m values shown are averages from three independent experiments. [See online article for color version of this figure.]

Figure 11.

Western-blot analysis of total proteins from Chlamydomonas wild type (CC425), rep27 mutant, and rep27-comp, rep27-ΔCt, rep27-ΔT1, rep27-ΔT2, and rep27-ΔT1+2 transformant strains probed with specific polyclonal antibodies against REP27 (A) or D1 (B) proteins. Also shown is the Coomassie Brilliant Blue-stained SDS-PAGE profile of the proteins from the various samples (C). Four micrograms of chlorophyll was loaded onto 12% acrylamide for the SDS-PAGE and western-blot analyses.

Figure 12.

Schematic of the REP27 protein function in D1 protein turnover. The REP27 C terminus is essential for the de novo D1 biosynthesis at the level of ribosomal psbA mRNA translation and initial assembly in the PSII template. The TPR motifs participate in posttranslational modification (D1 activation). Both TPR motifs are needed to activate the nascent D1 and to confer functional status to the PSII holocomplex.