Lysine acetylation regulates moonlighting activity of the E2 subunit of the chloroplast pyruvate dehydrogenase complex in Chlamydomonas.

The dihydrolipoamide acetyltransferase subunit DLA2 of the chloroplast pyruvate dehydrogenase complex (cpPDC) in the green alga Chlamydomonas reinhardtii has previously been shown to possess moonlighting activity in chloroplast gene expression. Under mixotrophic growth conditions, DLA2 forms part of a ribonucleoprotein particle (RNP) with the psbA mRNA that encodes the D1 protein of the photosystem II (PSII) reaction center. Here, we report on the characterization of the molecular switch that regulates shuttling of DLA2 between its functions in carbon metabolism and D1 synthesis. Determination of RNA-binding affinities by microscale thermophoresis demonstrated that the E3-binding domain (E3BD) of DLA2 mediates psbA-specific RNA recognition. Analyses of cpPDC formation and activity, as well as RNP complex formation, showed that acetylation of a single lysine residue (K197) in E3BD induces the release of DLA2 from the cpPDC, and its functional shift towards RNA binding. Moreover, Förster resonance energy transfer microscopy revealed that psbA mRNA/DLA2 complexes localize around the chloroplast's pyrenoid. Pulse labeling and D1 re-accumulation after induced PSII degradation strongly suggest that DLA2 is important for D1 synthesis during de novo PSII biogenesis.