Hierarchy of Synthesis among PSII Core Subunits.
(A) Rate of synthesis of the major PSII subunits, determined by 5-min pulse-labeling experiments in wild-type and deletion strains of C. reinhardtii. Analysis on 8% acrylamide-urea gels. An asterisk marks a neosynthesized form of D1.
(B) Accumulation of psbB, psbD, psbA, and petA (loading control) transcripts in the same strains.

psbB mRNA Is Poorly Translated in the Absence of D1.
Left panels: Distribution of psbB, psbD, and atpB mRNAs on sucrose gradients in the wild type and in ΔpsbA and tba2-F35 mutants lacking expression of D1. The distribution of rrnS and psbB RNAs in extracts from wild-type cells, in conditions preserving polysomes (+CAP) or disrupting polysomes (+EDTA), is shown to substantiate the assignment of polysome and free subunits to specific fractions. Fraction P contains the material found as a pellet at the bottom of the tube. The black circle in panel ΔpsbA indicates RNA degradation in that specific fraction. Right panels: mRNA distributions were quantified by PhosphorImager scan of ³³P labeling bound to the membrane in the three strains and expressed as the percentage of total transcript found in each fraction.

Cytochrome f Reporter Driven by the psbB-5′UTR Is Overexpressed in the Absence of Both the Assembly Partner (D1) and the CES (CP47) Subunits.
(A) Chloroplast translation products in representative tetrad progeny from {CP47_Tr, bBf} × tba2-F35 crosses and in the wild-type and parental strains. Cells expressing only the chimeric gene 5′psbB-petA or the truncated psbB allele are shown for comparison. The black circles indicate the position of the truncated apoCP47.
(B) Accumulation of cytochrome f and OEE2 (loading control) in the same strains, detected using specific antibodies.
(C) Accumulation of petA, psbA, psbB, and atpA (loading control) transcripts in the same strains.

The CES Behavior of D1 Corresponds to a Translational Regulation Mediated by the psbA-5′UTR.
(A) Schematic representation of the chimeric gene 5′psbA-petA, using conventions of Figure 2A. The small rectangle upstream of the N* site depicts the first 60 nucleotides of the psbA coding sequence.
(B) Chloroplast translation products in a representative half tetrad of bAf × mbd1-nac2 crosses and in parental and wild-type strains.
(C) Accumulation of cytochrome f and OEE2 (loading control) in the same strains, immunodetected with specific antibodies.
(D) Accumulation of petA, psbD, psbA, and atpA (loading control) transcripts in the same strains.
(E) Expression of the chimeric gene 5′psbA-petA in the deletion strain ΔpsbD. The wild-type and bAf strains are shown as controls.

The D1_Tr Strain Expresses a Truncated and Short-Lived psbA Gene Product.
(A) Top: Map of the psbA gene. Exons are indicated by gray boxes. The bent arrow indicates transcription start site. Bottom: schematic enlarged view of the insert in p-157BS, were a stop codon (linked to a BglII restriction site) was introduced in the 5th exon of the psbA gene (in the IV to V connecting loop) and associated with the recycling spectinomycin resistance cassette, schematically depicted by the boxed K with chevrons on either side (not to scale). The sequence coding for the fifth transmembrane helix is shown as a white box. Relevant restriction sites are as follows: P, PstI; X, XbaI; A, ApaI; D, DraIII; S, SacII; B, BstEII; Bg, BglII; Ba, BamHI. The position of the mutation with respect to the protein sequence is indicated in the schematic structure below.
(B) Expression of the truncated D1 polypeptide: psbA translation products were analyzed by pulse-labeling experiments in the wild type, in a transformed strain expressing the truncated psbA allele D1_Tr, in a representative tetrad progeny from the cross D1_Tr × tba2-F35, and in the parental strain tba2-F35. Asterisks mark tetrad members carrying the tba2 mutation.
(C) Stability of the truncated protein assessed by pulse-chase experiments. Pulse-labeled wild type is shown for comparison. The positions of D2 and D1 and of the truncated D1 polypeptide are indicated.

The CES Process Does Not Take Part in the Recovery from Photoinhibition.
Wild type, bAf, {ΔpsbA, bAf} and ΔpsbD (A) or wild type, bA_2Hf, bAfbA and {ΔpsbB, bAf} strains (B) were pulse labeled under normal light (left) or photoinhibition conditions (right). Asterisks indicate the neosynthesized form of D1 described in Figure 1.

Expression of the 5′psbB-petA Chimeric Gene Decreases in the Absence of D1.
(A) Map of the chloroplast petA gene in wild-type and bBf strains. Relevant restriction sites are indicated: B, BglII; N*, an NcoI site engineered around the petA initiation codon for cloning purposes; H, HincII. The bent arrow indicates transcription start sites. K stands for the spectinomycin resistance cassette, in opposite orientation with respect to petA.
(B) Translation of the chimeric gene in a representative half tetrad from bBf × tba2 crosses and in parental and wild-type strains. Translation of the chimeric gene in the ΔpsbA chloroplast mutant is also shown.
(C) and (D) Protein (C) and transcript (D) accumulation of the chimeric gene in the same strains. Accumulation of OEE2 and of atpB transcript provide loading controls.

Characterization of a Strain Expressing a Truncated apoCP47 Polypeptide.
(A) Top line: Map of the psbB gene. Transmembrane helices are represented as gray boxes. The bent arrow indicates transcription start site. Bottom line: Schematic map of the construct used to introduce a stop codon (linked to a HindIII restriction site) into the psbB coding sequence as well as a recycling spectinomycin resistance cassette, schematically depicted by the boxed K with chevrons on either side (not to scale), for selection of transformants. Restriction sites used are as indicated: A, ApaLI; P, PvuII; S, StuI; H, HindIII. The position of the mutation with respect to the protein sequence is indicated below.
(B) Level of translation of the psbB mRNA (either endogenous or mutated) in the wild type, in the CP47_Tr strain expressing the truncated apoCP47 (marked by a black circle), in a representative tetrad progeny from the cross CP47_Tr × mbb1-222E, and in the parental strain mbb1-222E. Asterisks mark the tetrad members carrying the mbb1 mutation.
(C) Stability of the truncated apoCP47 polypeptide determined by pulse labeling of the CP47_Tr strain (time 0) followed by a chase for the indicated times in the presence of an excess of nonradioactive acetate and 200 μg·mL⁻¹ chloramphenicol. Pulse-labeled wild type is shown for comparison.

psbA mRNA Association with Polysomes
(A) Distribution of psaA (as a control) and psbA mRNAs on sucrose gradients in the wild-type and ΔpsbD strains. Fraction P contains the pellet, rinsed out from the bottom of the tube after collection of fractions 1 to 10.
(B) mRNA distribution was quantified from a PhosphorImager scan of the ³³P labeling and expressed as the percentage of total transcript found in each fraction. The bottom panel shows an enlarged view of the distribution of psbA mRNA in the heavy fractions.

The psbA 5′UTR Confers a D2-Dependent Expression to the Reporter Gene aadA.
(A) Schematic map of the petA-petD region where the 5′psbA-aadA chimeric gene was inserted in direct orientation with respect to the petA gene, at the neutral EcoRV (V) site. The bent arrows indicate transcription start sites. The small rectangle upstream of aadA coding sequence depicts the 60 nucleotides from the psbA coding sequence.
(B) Growth of four independent transformants, derived from the recipient strains listed on the left, in the presence of increasing concentrations of spectinomycin plus streptomycin.
(C) Accumulation of the chimeric aadA (and petA as a loading control) transcripts in the wild type and in one representative transformant for each genetic background presented in (B).

The 5′psbA-petA Reporter Gene Is Overtranslated in Strains Expressing Truncated D1.
(A) Chloroplast translation products in representative offspring from the cross {D1_Tr, bAf} × mbd1-nac2 in wild-type, bAf, D1_Tr, and parental strains. Lack of D2 synthesis indicates the mbd1 progeny. PhosphorImager quantification of cytochrome f synthesis, relative to that observed in the wild type (after normalization to the level of synthesis of the β-subunit from the ATP synthase complex to correct for variations in the efficiency of ¹⁴C incorporation) is indicated below the figure.
(B) Accumulation of cytochrome f and OEE2 (loading control) in these strains. Quantification of cytochrome f accumulation (relative to the wild type) is shown below the panel.
(C) Transcript accumulation for petA, psbA, psbD, and atpB (loading control) in the same strains.

Model of the CES Cascade Involved in PSII Biogenesis.