Synthesis of the D2 protein of photosystem II in Chlamydomonas is controlled by a high molecular mass complex containing the RNA stabilization factor Nac2 and the translational activator RBP40

Abstract

Gene expression in chloroplasts is regulated mainly at the posttranscriptional level. In the green alga Chlamydomonas reinhardtii, synthesis of the D2 protein (PsbD), which is the rate-determining subunit for the assembly of photosystem II, depends on the RNA stability factor Nac2. In addition, the RNA binding protein RBP40 has been implicated in translational control via a U-rich element in the 5' untranslated region (5'UTR) of the psbD mRNA. Here, we report the identification of the RBP40 gene based on mass spectrometric analysis of its purified product. Unexpectedly, this was found to be identical to the previously described RNA binding protein RB38, which had been suggested to be involved in the regulation of D1 protein synthesis. However, we show that RBP40 binds to the psbD 5'UTR in a Nac2-dependent fashion both in vitro and in vivo. Molecular characterization of RBP40 RNA interference lines confirmed that RBP40 specifically affects the initiation of D2 synthesis. Native polyacrylamide gel electrophoresis, coimmunoprecipitation, and sedimentation analyses revealed that Nac2 and RBP40 form parts of a complex of 550 kD that is displaced from the psbD mRNA prior to polysome assembly. Together, these data indicate that the processes of 5'UTR-mediated RNA stabilization and translation initiation are tightly coupled in Chlamydomonas.

Figure 1.

Isolation of RBP40. (A) Flow chart listing the steps used to purify RBP40. (B) SDS-PAGE and Coomassie blue staining of proteins at various stages of purification (top panel), UV cross-linking of RBP40 to radiolabeled psbD 5′UTR RNA (middle panel), and immunodetection of Nac2 in selected fractions by protein gel blot analysis (bottom panel). neg., negative control for RNA binding (no protein loaded); cp, chloroplast lysate; S, stromal protein fraction; HepFT, flow-through fraction from heparin-Sepharose column; HepE150, -500, and -1000, eluates obtained with 150, 500, and 1000 mM KCl, respectively; pUFT/W, flow-through/wash fraction from poly(U)-Sepharose column; pUE150, -500, and -1000, eluates obtained with 150, 500, and 1000 mM KCl, respectively. The RBP40 that eluted with high salt from poly(U)-Sepharose is marked by the arrow.

Figure 2.

Nac2 Confers RNA Binding Specificity on RBP40. (A) The pUE1000 fraction containing the purified RBP40 (see Figure 1A) alone, in combination with the wild-type flow-through fraction from the poly(U)-Sepharose column (pUE+WTFT) containing Nac2, or with the same fraction from the mutant nac2 (pUE+nac2FT) was incubated with radiolabeled psbD 5′UTR RNA in the presence of a 5-, 50-, or 500-fold excess of the indicated competitor RNA and analyzed by UV cross-linking. (B) In the graphs, the intensities of the RBP40 signals are plotted against the relative levels of the indicated competitor RNAs in the reactions, based on densitometric scanning of the autoradiograms shown in (A).

Figure 3.

Coimmunoprecipitation of RBP40 and psbD mRNA. Chloroplast stromal proteins were used for immunoprecipitation reactions with an αRBP40 antiserum or the preimmune serum (PRE). RNAs were extracted from precipitates (αRBP40 and PRE) and supernatants (SαRBP40 and SPRE), and equal proportions were subjected to dot-blot hybridization using the radiolabeled DNA probes indicated at top.

Figure 4.

Molecular Characterization of RBP40 RNAi Lines. (A) Protein gel blot analysis of total proteins (10 μg) isolated from the indicated RBP40 RNAi lines and the wild type was performed using antibodies raised against the proteins indicated at left. (B) RNA gel blot analysis of psbD mRNA accumulation in RBP40 RNAi lines. (C) Total proteins from the indicated strains were pulse-labeled for 20 min with [³⁵S]sulfate and subsequently fractionated by SDS-PAGE. The positions of cytochrome f, D2, and D1 proteins are indicated (Klinkert et al., 2006). Fluctuations of signal intensities marked with asterisks were not seen reproducibly.

Figure 5.

Polysomal Loading of psbD mRNA. Whole-cell extracts from the wild type (WT+MgCl₂) and the RNAi line 40-9 (40-9+MgCl₂) were fractionated on 15 to 40% sucrose gradients by ultracentrifugation. As a negative control, polysomes were destabilized by the addition of EDTA (WT+EDTA). At the top of each of the three panels, the ethidium bromide–stained rRNA patterns before blotting of the gels are shown. Below, hybridization signals are shown that were obtained with the radiolabeled probes indicated at right. The sedimentation behavior of RBP40 in the wild type was followed by protein gel blot analysis of proteins from the same gradient fractions.

Figure 6.

Association of RBP40 with Ribosomes. Dot-blot hybridization with radiolabeled 16S and 23S rDNA probes was performed on immunoprecipitates similar to those shown in Figure 3.

Figure 7.

Native PAGE of RBP40 and Nac2. Stromal protein fractions from wild-type and nac2 chloroplasts were subjected to native PAGE on 8% gels and transferred to nitrocellulose filters, which were immunolabeled with either αNac2 or αRBP40 antibodies. The arrow indicates the 550-kD Nac2/RBP40 complex. Equal loading was confirmed by Ponceau red staining of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). The sizes of marker proteins are given at left.

Figure 8.

Coimmunoprecipitation of RBP40 and Nac2. Stromal protein fractions from wild-type and nac2 chloroplasts were incubated with either αRBP40 antibody coupled to protein A–Sepharose (IP αRBP40) (A) or αNac2 antibody coupled to protein G–Sepharose (IP αNac2) (B). After elution from the matrix, the material was subjected to SDS-PAGE and immunolabeled using the same antibodies (ID αNac2 or ID αRBP40). The asterisk marks material that cross-reacts with the αNac2 antiserum.

Figure 9.

Formation of an RBP40/Nac2 Complex in the Absence of psbD mRNA. Stromal chloroplast proteins from the strains indicated at left were centrifuged through 15 to 35% glycerol gradients. The distribution of the Nac2 and RBP40 proteins after centrifugation (at right) was monitored by protein gel blot analysis of the fractions marked below each filter strip. RBP40ee represents an extended exposure (>100-fold) of the blot shown directly above it.

Figure 10.

Working Model for the Posttranscriptional Control of psbD Gene Expression. The sequence of the psbD 5′UTR from Chlamydomonas is given with the PRB2 site, the U-rich translational element boxed in gray, and the putative Shine-Dalgarno element (PRB1) (Nickelsen et al., 1999). The AUG start codon (gray letters) is marked by Met. Putative additional components of the Nac2/RBP40 complex are indicated by a question mark. The closed arrow represents the change in RNA conformation induced by RBP40, and the open arrows stand for the subsequent binding of components of the translational machinery. For further explanation, see text.