The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability

Abstract

The psbD mRNA, which encodes the D2 reaction center polypeptide of photosystem II, is one of the most abundant chloroplast mRNAs. We have used genomic complementation to isolate the nuclear Nac2 gene, which is required for the stable accumulation of the psbD mRNA in Chlamydomonas reinhardtii. Nac2 encodes a hydrophilic polypeptide of 1385 amino acids with nine tetratricopeptide-like repeats (TPRs) in its C-terminal half. Cell fractionation studies indicate that the Nac2 protein is localized in the stromal compartment of the chloroplast. It is part of a high molecular weight complex that is associated with non-polysomal RNA. Change of a conserved alanine residue of the fourth TPR motif by site-directed mutagenesis leads to aggregation of Nac2 protein and completely abrogates its function, indicating that this TPR is important for proper folding of the protein and for psbD mRNA stability, processing and/or translation.

Fig. 1. Nac2 gene organization and expression. (A) Scheme of the 3′ region of the Nac2 cDNA with the coding region boxed. Total DNA from the wild-type (WT), nac2-26 and mø14 strains was digested with BglI restriction endonuclease, electrophoresed, transferred to nylon membranes and hybridized with the labeled probes A, B and C, the position of which is indicated on the cDNA map. The arrows represent the position of the primers used to synthesize probe A. The estimated sizes of the DNA fragments are indicated in kb. (B) RNA blot analysis of the wild-type, nac2-26 and mø14 strains. A 3 µg aliquot of poly(A) RNA from wild-type (WT), nac2-26 and mø14 was fractionated on a 1% agarose–formaldehyde gel, blotted to a nitrocellulose membrane and hybridized with the ³²P-labeled Nac2 cDNA probes A and C. Equal loading of mRNA was checked by hybridization of the blots with a rbcS probe. The sizes of the RNA species are indicated in kb and were determined by comparison with size markers. (C) DNA blot analysis of four transformants T1, T2, T3 and T4 obtained by transformation of the nac2-26 strain with the Nac2 cDNA cnac1 encoding the 588 C-terminal residues of Nac2. Probe B was used for the hybridization.

Fig. 2. Nac2 polypeptide. (A) Sequence of the predicted Nac2 polypeptide. The nine TPR-like repeats are highlighted in gray; dots indicate boundaries between the repeats. The mutated A in the fourth TPR repeat is underlined. The stippled line marks the putative transit sequence. The GTP/ATP-binding site is double underlined. Arrowheads indicate the location of introns. (B) Schematic view of the Nac2 polypeptide. The nine TPR-like repeats are indicated by boxes; G, ATP/GTP-binding site; the upward arrow marks the end of the C-terminal part of the Nac2 protein which is able to complement the Nac2 deficiency; the asterisk marks the site of the mutation. (C) Alignment of the nine TPR motifs of the Nac2 protein. Residues which appear at least five times amongst the nine TPRs are highlighted. The conserved residues of the Nac2 TPRs are shown at the bottom together with the TPR consensus at positions 4, 7, 8, 11, 20, 24, 27 and 32 (Lamb et al., 1995); h, hydrophobic residue; A and B, TPR α-helical domains A and B (Das et al., 1998).

Fig. 3. Immunodetection of the Nac2 protein. (A) Immunoblot analysis of total cell proteins (75 µg) of the wild-type (WT), cw15, nac2-26 and mø14. Total soluble and membrane fractions were used. The blots were reacted with polyclonal antiserum against the 40 kDa C-terminal part of the Nac2 protein. Molecular mass markers are indicated in kDa. (B) Subcellular localization of the Nac2 protein. Immunoblot analysis of total cell proteins from the wild-type (WT), mø14, nac2::HA, chloroplasts from nac2::HA (chlp), soluble chloroplast fraction from nac2::HA (sol) and chloroplast membrane fraction from nac2::HA (mem). The blots were reacted with anti-HA monoclonal antibody and antibodies against the large subunit of Rubisco (RbcL), PsaA and eIF4A. The proteins reacting with the eIF4A antibody in the chloroplast lane could represent a plastid form of a factor related to eIF4A. Molecular mass markers are indicated in kDa.

Fig. 4. The Nac2 protein is part of a high molecular weight complex. Soluble cell extracts were prepared as described in Materials and methods. (A) A soluble cell extract from mø14 transformed with Nac2::HA was centrifuged on a 0.3–1.3 M linear sucrose gradient for 21 h at 230 000 g. Sedimentation was from left to right. Aliquots of the fractions were electrophoresed on a SDS–6% polyacrylamide gel, immunoblotted and reacted with anti-HA monoclonal antibody. (B) The soluble cell extracts from mø14 transformed with wild-type Nac2::HA (rows1–3) or mutant Nac2 A₁₀₃₈E::HA (rows 4–6) were concentrated to a final concentration of 20 µg/ml and fractionated on a Superose 6 PC3.2/30 column using the SMART system (Pharmacia Biotech, Sweden). Twenty-four fractions were collected and aliquots were electrophoresed on a 6% SDS–polyacrylamide gel and reacted with anti-HA monoclonal antibody (rows 1–6) and antibodies against RB60 (rows 7 and 8) and RbcL (rows 9 and 10). Sizes in each fraction of (A) and (B) were determined by comparison with size standards included in the HMW Gel Filtration Calibration kit (Pharmacia Biotech, Sweden). (C) Total RNA was extracted from 28 fractions collected after size exclusion chromatography with extracts from the nac2::HA strain. RNAs were stained with ethidium bromide and electrophoresed through an agarose–formaldehyde denaturing gel and visualized under UV light. (D) RNA from each fraction was slot-blotted and hybridized with a ³²P-labeled psbD probe.

Fig. 5. The Nac2 protein is not associated with polysomes. Polysomes were prepared as described in Materials and methods. (A) Immunoblot analysis of the supernatant and polysome fractions from the nac2::HA strain. Samples were loaded on a 6% SDS–polyacrylamide gel and reacted with anti-HA monoclonal antibody. Molecular mass markers are indicated in kDa. (B) RNA content of the supernatant and polysome fractions from the nac2::HA strain. Total RNA was isolated by phenol/chloroform extraction followed by ethanol precipitation. The samples were stained with ethidium bromide and electrophoresed on an agarose–formaldehyde denaturing gel. RNA sizes are indicated in kb.

Fig. 6. The Nac2 A₁₀₃₈E protein is expressed but does not rescue the mø14 null mutant. (A) Immunoblot analysis of total cell proteins from the nac2::HA, mø14 and nac2 A₁₀₃₈E::HA strains. Serial dilutions of the nac2::HA extracts were performed with total cell extracts from the mø14 strain in order to maintain the protein amount (100 µg) constant in each lane. Samples were loaded on a 6% SDS–polyacrylamide gel and reacted with anti-HA monoclonal antibody. (B) Fluorescence transients of dark-adapted cells of the wild-type (WT), mø14, nac2::HA and nac2 A₁₀₃₈E::HA strains. Cells were grown in liquid TAP medium under dim light and dark-adapted for 1 min before measurements.