Figure 8.

LSU₂ mutations alter LSU accumulation and CES regulation. (A) Close-up of the mutated residues in LSU₂mut strain in LSU structure. C. reinhardtii LSU dimer structure is shown in cartoon, as extracted from Rubisco structure (PDB: 1IR2). The two LSU subunits forming the dimer are represented in green and magenta. Subunits are maintained by two inter-subunits salt bridges between E109 and R253, and E110 and R213 residues. Residues mutated in LSU₂mut (E109A and R253A) are highlighted in red. The figure was generated using the PyMol program (Schrödinger-LLC). (B) Impairment in Rubisco accumulation is revealed by the absence of phototrophic growth in the LSU₂mut and ΔRBCS;LSU₂mut strains as probed by spot tests on acetate-free minimal media (MIN). Growth on TAP is shown as a control. The corresponding soluble LSU accumulation detected by immunoblot is shown together with PsaD accumulation as loading control. (C) LSU synthesis rate in LSU₂mut and ΔRBCS;LSU₂mut measured by short ¹⁴C pulse labeling experiment and compared to WT. Note that in the 12–18% acrylamide-8M urea gel system, the mutated LSU undergoes a change in its migration pattern compared to native LSU. (D) Immunoblot with the Rubisco antibody after CN-PAGE analysis of soluble protein fractions of WT (note the dilution), ΔrbcL, ΔRBCS, LSU₂mut and ΔRBCS;LSU₂mut strains. A dashed line marks the position where two irrelevant lanes were removed. The position of the LSU-complexes observed in ΔRBCS is indicated at the right using the same symbols as in Figure 5 (square, cross, and circle).