Translation of cytochrome f is autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas chloroplasts

Abstract

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5' untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the atpA 5' untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric petA-aadA-rbcL gene was shown to depend on the state of assembly of cytochrome b6f complexes and on the accumulation of the C-terminal domain of cytochrome f. We discuss the possible ubiquity of the CES process in organellar protein biogenesis.

Figure 1

Synthesis of hemeless cytochrome f escapes the CES process. Chloroplast translates from wild-type, ΔpetD, F52L-55V, and F52L-55VΔpetD strains (lanes from left to right). Whole-cell polypeptides were pulse-labeled for 5 min with [¹⁴C]acetate in the presence of an inhibitor of cytoplasmic translation and separated in SDS/12–18% polyacrylamide gels in the presence of 8 M urea. Arrowhead, positions of cytochrome f and SUIV deduced from comparison with known polypeptide patterns; ∗, position of apocytochrome f, which migrates slightly faster than holocytochrome f (20).

Figure 2

Cytochrome f synthesis under control of the atpA 5′ UTR escapes the CES process. (A) Maps of the petA gene in wild-type and AFFF strains (Bg, BglII; N, NcoI). The heavily hatched box in the 5′ region of atpA denotes the sequence encoding the 25 first amino acids of the α subunit of the ATP synthase complex. (B) Newly synthesized cytochrome f and SUIV detected by pulse-labeling experiments in ΔpetA, wild-type, parental strains, and progeny of a representative tetrad from the cross AFFF × mcd1-F16. (C) Accumulation of cytochromes f and b₆, detected with specific antibodies in the same strains.

Figure 3

petA 5′ UTR confers CES behavior to the aadA reporter gene product. (A) Map of the R12 fragment in wild-type and FKR12 strains. The positions of known genes and relevant restriction sites are indicated (S, StuI; RI, EcoRI) (31). (B) Percentage of strains with FKR12/mcd1, FKR12/WT, and FKR12/ccs1 genotypes, as indicated, growing on TAP (T0), T1, T2, or T3 medium, as indicated. Absolute numbers of growing clones (resistant clones) are indicated in C. The 62 FKR12/WT strains originated from three crosses—32 from FKR12 × WT, 14 from FKR12 × mcd1-F16, and 16 from FKR12 × ccs1-ac206—that yielded the same ratio of about 50% resistant clones on T2.

Figure 4

Hypothetical mechanisms for the CES behavior of cytochrome f. (A) Direct interaction between the C_ter domain of cytochrome f and the petA 5′ UTR. (B) Indirect interaction which relies on a putative ternary effector, TCA_i, an activator of cytochrome f translation. (Left) Inhibition of cytochrome f synthesis caused by the accumulation of unassembled cytochrome f, as observed in strains lacking SUIV, but not in strains lacking accumulation of the C_ter domain of cytochrome f. (Right) Activation of cytochrome f synthesis, the petA messenger (A) or the TCA_i factor (B) being made available upon assembly. The intermediate rate of synthesis observed in wild-type cells results from an equilibrium between these situations.