C172S Substitution in the Chloroplast-encoded Large Subunit Affects Stability and Stress-induced Turnover of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

Joaquín Moreno,Robert J Spreitzer

doi:10.1074/jbc.274.38.26789

Joaquín Moreno, Robert J Spreitzer

Open Access

https://doi.org/10.1074/jbc.274.38.26789

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Abstract

Previous work has indicated that the turnover of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1. 39) may be controlled by the redox state of certain cysteine residues. To test this hypothesis, directed mutagenesis and chloroplast transformation were employed to create a C172S substitution in the Rubisco large subunit of the green alga Chlamydomonas reinhardtii. The C172S mutant strain was not substantially different from the wild type with respect to growth rate, and the purified mutant enzyme had a normal circular dichroism spectrum. However, the mutant enzyme was inactivated faster than the wild-type enzyme at 40 and 50 degrees C. In contrast, C172S mutant Rubisco was more resistant to sodium arsenite, which reacts with vicinal dithiols. The effect of arsenite may be directed to the cysteine 172/192 pair that is present in the wild-type enzyme, but absent in the mutant enzyme. The mutant enzyme was also more resistant to proteinase K in vitro at low redox potential. Furthermore, oxidative (hydrogen peroxide) or osmotic (mannitol) stress-induced degradation of Rubisco in vivo was delayed in C172S mutant cells relative to wild-type cells. Thus, cysteine residues could play a role in regulating the degradation of Rubisco under in vivo stress conditions.

Highlights

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco1; EC 4.1.1.39) catalyzes the photosynthetic fixation of CO2 through the Calvin cycle, the metabolic route that accounts for most of the carbon input into the biosphere
Because the difference between the velocities of carboxylation and oxygenation determines net photosynthetic CO2 fixation [1, 8], many studies have been devoted to understanding the structural basis for CO2/O2 specificity by screening for chloroplast Rubisco mutants in the green alga Chlamydomonas reinhardtii or by expressing directed mutant prokaryotic enzymes in Escherichia coli
Proteolysis of Rubisco is a prominent feature in the stress responses of plants and algae, and several studies have shown that Rubisco is oxidatively modified prior to its degradation [15,16,17,18,19,20]

Summary

EXPERIMENTAL PROCEDURES

Strains and Culture Conditions—C. reinhardtii 2137 mtϩ is the wild-type strain [29]. Rubisco mutant 18-7G mtϩ was used as the host for transformation. The plasmid containing the C172S rbcL mutant gene was named pLS-C172S Chloroplast transformation with this plasmid was performed via microprojectile bombardment [34] as described previously [10, 13]. Redox Buffers and Susceptibility to Proteolysis—Aliquots (200 ␮l) of purified Rubisco (0.2 mg/ml) were combined with 100 ␮l of cystamine/ cysteamine mixtures of constant monomeric concentration (120 mM in 100 mM Tris-HCl, pH 8.2, 10 mM MgCl2, and 10 mM NaHCO3). In Vivo Stress Experiments—Rubisco content was determined by centrifuging 1 ml of culture at 5000 ϫ g for 1 min; resuspending the pellet in 0.2 ml of 83 mM Tris-HCl, pH 6.8, 0.66 M 2-mercaptoethanol, 2.17% SDS, and 8.3% (w/v) glycerol; and boiling for 4 min. The samples were quantified by blot densitometry [17, 22]

RESULTS

DISCUSSION

RuBP carboxylase activitya