Dynamic light‐ and acetate‐dependent regulation of the proteome and lysine acetylome of Chlamydomonas

Abstract

SUMMARY The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post‐translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ‐amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry‐based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin–Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

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

“…This indeed suggests that the increased acetylation of K197 under mixotrophic growth conditions observed by Füßl et al. (2022) might lead to an increase in membrane‐associated DLA2. Importantly, the simulation of constitutive acetylation of K197 in the K197Q‐HA strain enhanced the amounts of DLA2 associated with the membrane under both growth conditions (Figure 5b).…”

“…The higher levels of DLA2 detected in RNPs under mixotrophic conditions cannot be attributed to the availability of larger amounts of DLA2, as equal quantities of the protein were observed under photoautotrophic and mixotrophic growth conditions (Bohne et al., 2013b; Füßl et al., 2022). However, the fact that RNA‐binding of DLA2 is dependent on acetate pointed to a potential role for post‐translational protein acetylation events.…”

“…Indeed, a recent analysis of the lysine acetylome of Chlamydomonas by Füßl et al. (2022) identified eight lysine acetylation sites in DLA2, i.e., at positions K56, K61, K119, K120, K197, K306, K315, and K394 (Figure S1). Of these, only the acetylation of K197 was significantly and strongly increased 4‐fold (adjusted P ‐value < 0.02) under mixo‐ and heterotrophic conditions compared to photoautotrophic growth, which lacked external acetate.…”

“…Accordingly, K197 in DLA2 was replaced by either glutamine (K197Q) or arginine (K197R). Notably, in addition to K197, the E3BD contains two more lysine residues – K193 and K200 – that were not found to be acetylated in vivo (Füßl et al., 2022). These two lysines were investigated in parallel to serve as controls.…”

Light-dependence of Formate (C1) and Acetate (C2) Transport and Oxidation in Poplar Trees

“…Similarly, in the case where acetate is activated to acetyl-CoA within peroxisomes as a part of the glyoxylate cycle, peroxisomal CS may also be strongly dark activated. Recent studies with the photosynthetic green alga Chlamydomonas reinhardtii demonstrate that not only do the abundances of enzymes involved in the glyoxylate cycle become elevated under heterotrophic conditions (dark and in the presence of external acetate), peroxisomal CS activity dramatically increases via protein acetylation [51]. Nonetheless, it is important to note that other mechanisms could also be at play, and lead to a similar "apparent" suppression of [ 13 C 2 ]acetate respiration in the light including the re-assimilation of respiratory 13 CO 2 by photosynthesis [12].…”