Targeted quantitative analysis of a diurnal RuBisCO subunit expression and translation profile in Chlamydomonas reinhardtii introducing a novel Mass Western approach

Abstract

RuBisCO catalyzes the rate-limiting step of CO2 fixation in photosynthesis. Hypothetical mechanisms for the regulation of rbcL and rbcS gene expression assume that both large (LSU) and small (SSU) RuBisCO subunit proteins (RSUs) are present in equimolar amounts to fit the 1:1 subunit stoichiometry of the holoenzyme. However, the actual quantities of the RSUs have never been determined in any photosynthetic organism. In this study the absolute amount of rbc transcripts and RSUs was quantified in Chlamydomonas reinhardtii grown during a diurnal light/dark cycle. A novel approach utilizing more reliable protein stoichiometry quantification is introduced. The rbcL:rbcS transcript and protein ratios were both 5:1 on average during the diurnal time course, indicating that SSU is the limiting factor for the assembly of the holoenzyme. The oscillation of the RSUs was 9h out of phase relative to the transcripts. The amount of rbc transcripts was at its maximum in the dark while that of RSUs was at its maximum in the light phase suggesting that translation of the rbc transcripts is activated by light as previously hypothesized. A possible post-translational regulation that might be involved in the accumulation of a 37-kDa N-terminal LSU fragment during the light phase is discussed.

Biological significance: A novel MS based approach enabling the exact stoichiometric analysis and absolute quantification of protein complexes is presented in this article. The application of this method revealed new insights in RuBisCO subunit dynamics.

Keywords: Chlamydomonas reinhardtii; Mass Western; Protein complex stoichiometry; RuBisCO; SRM; Targeted protein absolute quantification.