The (C)F1 sector from H(+)-ATP synthases comprises five subunits: alpha, beta, gamma, delta and epsilon, assembled in a 3:3:1:1:1 stoichiometry. Here, we describe the molecular mechanism ensuring this unique stoichiometry, required for the functional assembly of the chloroplast enzyme. It relies on a translational feedback loop operating in two steps along the assembly pathway of CF1. In Chlamydomonas, production of the nucleus-encoded subunit gamma is required for sustained translation of the chloroplast-encoded subunit beta, which in turn stimulates the expression of the chloroplast-encoded subunit alpha. Translational downregulation of subunits beta or alpha, when not assembled, is born by the 5'UTRs of their own mRNAs, pointing to a regulation of translation initiation. We show that subunit gamma, by assembling with alpha(3)beta(3) hexamers, releases a negative feedback exerted by alpha/beta assembly intermediates on translation of subunit beta. Moreover, translation of subunit alpha is transactivated by subunit beta, an observation unprecedented in the biogenesis of organelle proteins.

RIP (repeat‐induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C‐G to T‐A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP‐like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating‐type region. The 9 kb sequenced show 106 C‐G to T‐A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP‐like event extended to single‐copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C‐methylation. Interestingly, the N. crassa zeta–eta sequence, which acts as a potent de novo C‐methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.

Studies of the biogenesis of the photosynthetic protein complexes in the unicellular green alga Chlamydomonas reinhardtii have pointed to the importance of the concerted expression of nuclear and chloroplast genomes. The accumulation of chloroplast- and nuclear-encoded subunits is concerted, most often as a result of the rapid proteolytic disposal of unassembled subunits, but the rate of synthesis of some chloroplast-encoded subunits from photosynthetic protein complexes, designed as CES proteins (Controlled by Epistasy of Synthesis), is regulated by the availability of their assembly partners from the same complex. Cytochrome f, a major subunit of the cytochrome b(6)f complex is a model protein for the study of the CES process. In the absence of subunit IV, another subunit of the cytochrome b(6)f complex, its synthesis is decreased by 90%. This results from a negative autoregulation of cytochrome f translation initiation, mediated by a regulatory motif carried by the C-terminal domain of the unassembled protein [Choquet, Stern, Wostrikoff, Kuras, Girard-Bascou and Wollman (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4380-4385]. Using site-directed mutagenesis, we have characterized this regulatory motif. We discuss the possible implications regarding the mechanism of the CES process for cytochrome f expression. We have studied the possible generalization of this mechanism to other CES proteins.