Translation of the chloroplast psbA mRNA requires the nuclear-encoded poly(A)-binding protein, RB47

Abstract

A set of nuclear mutants of C. reinhardtii were identified that specifically lack translation of the chloroplast-encoded psbA mRNA, which encodes the photosystem II reaction center polypeptide D1. Two of these mutants are deficient in the 47-kD member (RB47) of the psbA RNA-binding complex, which has previously been identified both genetically and biochemically as a putative translational activator of the chloroplast psbA mRNA. RB47 is a member of the poly(A)-binding protein family, and binds with high affinity and specificity to the 5' untranslated region of the psbA mRNA. The results presented here confirm RB47's role as a message-specific translational activator in the chloroplast, and bring together genetic and biochemical data to form a cohesive model for light- activated translational regulation in the chloroplast.

Figure 1

Protein accumulation in wt and mutant strains. Equal quantities of membrane or soluble proteins were separated by SDS-PAGE and blotted to nitrocellulose. Identical filters were probed with antiserum against D1, D2, LHCII, ATPase, ribulose bis-phosphate oxygenase/ carboxylase, and a component of the PS II OEE1. Representative immunoblots of proteins from nuclear mutant hf149 compared with wt and Fud7, a chloroplast mutant with a psbA gene deletion (4).

Figure 2

Synthesis of chloroplast-encoded proteins from wt and hf149 mutant strains measured by [¹⁴C]acetate pulse labeling. Equal quantities of labeled proteins were loaded in each lane. The positions of the major components of PS II (D1, D2, P5, and P6) as well as ATPase are indicated.

Figure 3

Northern blot analysis of psbA (A and C) and psbD (B) mRNA accumulation in wt and mutant strains. Total RNA and RNA associated with polysomes was examined for the presence of these RNAs. 2 μg of RNA was loaded in each lane. (A and B) Total and polysomal RNA from the hf149 mutant strain probed with labeled psbA (A) or psbD (B) cDNAs. Dilutions of wt total RNA are shown for comparison to levels of RNA in the hf149 mutant strain. (C) Levels of total and polysomal psbA RNA are shown for the hf233, hf261, hf859, and hf1085 mutant strains.

Figure 3

Northern blot analysis of psbA (A and C) and psbD (B) mRNA accumulation in wt and mutant strains. Total RNA and RNA associated with polysomes was examined for the presence of these RNAs. 2 μg of RNA was loaded in each lane. (A and B) Total and polysomal RNA from the hf149 mutant strain probed with labeled psbA (A) or psbD (B) cDNAs. Dilutions of wt total RNA are shown for comparison to levels of RNA in the hf149 mutant strain. (C) Levels of total and polysomal psbA RNA are shown for the hf233, hf261, hf859, and hf1085 mutant strains.

Figure 4

psbA RNA binding activity in wt and mutant strains. Binding activity with 6 μg of protein was measured by T1-GMS assays. The first lane in each panel contains labeled RNA but no proteins (no lysate) for comparison.

Figure 5

Protein accumulation of psbA-specific RNA-binding proteins RB60 and RB38 in wt and mutant strains. Antisera against RB60 (A) and RB38 (B) were used for immunoblots of two identical filters with 3 μg of heparin agarose-purified proteins loaded in each lane. Proteins were separated by SDS-PAGE and blotted to nitrocellulose filters before decoration with specific antisera.

Figure 6

Protein accumulation of psbA specific RNA-binding protein RB47 in wt and mutant strains. Left panel, Coomassie-stained gel of equal amounts of wt and hf149 heparin agarose- purified proteins. Molecular weight standards are indicated at the left. Middle panel, antiserum against RB47 was used for an immunoblot of a gel with lanes identical to the left panel as well as additional lanes with threefold excess (3×) protein loaded for both wt and hf149. Right panel, immunoblot using antiserum against RB47 on heparin agarose-purified proteins from wt and hf233, hf261, hf859, and hf1085 mutant strains. A dilution of wt is shown for comparison to the mutant strains.

Figure 7

A model for light-activated translational regulation of the psbA mRNA in the chloroplast. A reducing environment is generated by PSII during the light reactions of photosynthesis. These reducing equivalents are passed through photosystem I (PSI), ferrodoxin (FD), and ferrodoxin-thioredoxin reductase (FDTR) to the chloroplast PDI (RB60). RB60 modulates the binding of RB47 to the psbA mRNA in a redox dependent manner through reversible oxidation of sulfhydryl groups in RB47. Binding of RB47 to the structured 5′ UTR of the psbA mRNA is required for translation of the mRNA. The translational activator RB47 acts concurrently to target the psbA mRNA to the membrane, most likely by facilitating translation initiation on membrane (mb−) bound ribosomes. Once the D1 protein is translated, it can be readily inserted into the membrane to assemble into PSII complexes.