Potential for interactions between the carboxy‐ and amino‐termini of Rubisco activase subunits

Abstract

The subunit interactions of Rubisco activase were investigated using mutants containing an introduced Cys near the N-and/or C-terminus. Chemical cross-linking of the C-terminal and double insertion mutant produced subunit dimers and dimers plus high ordered oligomers, respectively. Fluorescence measurements with N,NP P-dimethyl-N-(iodoacetyl)-NP P-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine showed that the environment around the introduced Cys near the C-terminus becomes more hydrophilic upon nucleotide binding. The Cys insertion mutants catalyzed Rubisco activation and ATP hydrolysis even when the subunits of the C-terminal or double insertion mutants were completely cross-linked. The results indicate that the termini of adjacent activase subunits are in close proximity and can be modi¢ed and even joined without a¡ecting enzyme function.

Increased Sensitivity of Oxidized Large Isoform of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase to ADP Inhibition Is Due to an Interaction between Its Carboxyl Extension and Nucleotide-binding Pocket

“…Moreover, cross-linking of the mutants enhanced their activity. However, no cross-linking was observed between the mutants and the wild type large isoform (24).…”

“…A new cysteine residue was inserted at position 402 (C402 INS ). Protein Expression and Purification-The recombinant activases and thioredoxin-f were expressed and purified as reported previously (13,24). Isolation of native Rubisco was performed as reported previously (26).…”

“…smaller isoform and exploits the observation that the endogenous cysteine residues (outside of the C-extension) are not very reactive (7,24). In the absence of ADP, the C402 INS mutant had ATP hydrolysis and Rubisco activation activities comparable with those of wild type 46-kDa activase ( Table 2).…”

“…A connection between redox regulation of activase and monomer-oligomer exchange was proposed, based on the fact that the altered sensitivity to the ADP/ATP ratio of the large isoform via redox treatments is sufficient to regulate the activities of both isoforms when mixed at a 1:1 ratio, although the activity of the smaller isoform itself is not altered by redox treatments (23). Recently, an effort to better understand the subunit interactions of activase was made by using mutants containing an introduced cysteine(s) near the N and/or C terminus of the small isoform of cotton activase and homobifunctional sulfhydryl-reactive cross-linkers (24). The N and C termini of the small isoform, which is 40 amino acids shorter than the large isoform in cotton, were shown to be in close proximity.…”

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

“…34 As described previously, a fluorescent label was attached to the Rca C-terminus by employing a carboxyterminal Ala-Cys insertion variant originally derived from Gossypium hirsutum (cotton) short-form (β) Rca. 42 Here, this variant is termed β-Rca-AC and is also referred to as wild-type protein (loosely defined). This protein was expressed in E. coli as an N-terminally 6His-tagged fusion protein and purified by Ni-affinity chromatography.…”