The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the   ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Towards that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N-terminus of the Rubisco large subunit. Large subunits lacking residues 1-4 are functional Rubiscos, but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model where Rca transiently threads the Rubisco large subunit N-terminus through the axial pore of the AAA+ hexamer.

During photosynthesis the AAA+ protein and essential molecular chaperone Rubisco activase (Rca) constantly remodels inhibited active sites of the CO2-fixing enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) to release tightly bound sugar phosphates. Higher plant Rca is a crop improvement target, but its mechanism remains poorly understood. Here we used structure-guided mutagenesis to probe the Rubisco-interacting surface of rice Rca. Mutations in Ser-23, Lys-148, and Arg-321 uncoupled adenosine triphosphatase and Rca activity, implicating them in the Rubisco interaction. Mutant doping experiments were used to evaluate a suite of known Rubisco-interacting residues for relative importance in the context of the functional hexamer. Hexamers containing some subunits that lack the Rubisco-interacting N-terminal domain displayed a ∼2-fold increase in Rca function. Overall Rubisco-interacting residues located toward the rim of the hexamer were found to be less critical to Rca function than those positioned toward the axial pore. Rca is a key regulator of the rate-limiting CO2-fixing reactions of photosynthesis. A detailed functional understanding will assist the ongoing endeavors to enhance crop CO2 assimilation rate, growth, and yield.

The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms inhibited complexes with multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. At least three classes of ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas) have evolved to remodel inhibited Rubisco complexes. The mechanism of green-type Rca found in higher plants has proved elusive, because until recently higher plant Rubiscos could not be expressed recombinantly. Towards identifying interaction sites between Rubisco and Rca, here we produce and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. We find that Rca activity is highly sensitive to truncations and mutations in the conserved N-terminus of the Rubisco large subunit. Both T5A and T7A substitutions cannot be activated by Rca, but present with increased carboxylation velocities. Our results are consistent with a model where Rca functions by transiently threading the Rubisco large subunit N-terminus through the axial pore of the AAA+ hexamer.

2PG 2-Phosphoglycolate 35S 35S promoter derived from the Cauliflower mosaic virus 3PG 3-phosphoglycerate A.thaliana / At Arabidopsis thaliana A.tumefaciens Agrobacterium tumefaciens AAA+ ATPase associated with various cellular activities ABRC Arabidopsis Biological Resource Center ADP Adenosine diphosphate AfM Acidithiobacillus ferrooxidans Form II Rubisco Amp Ampicilin ANOVA Analysis of variance ATP Adenosine Triphosphate ATPase ATP hydrolysis ATPyS Adenosine-5'-(γ-thio)-triphosphate BASTA Glufosinate-ammonium Bsd2 Bundle Sheath Defective 2 C.reinhartii / Cr Chlamydomonas reinhartii CA1P 2-Carboxy-arabinitol 1-phosphate CABP 2-Carboxy-arabinitol-1,5-bisphosphate CAM Crassulacean acid metabolism CAMV Cauliflower mosaic virus CBB Calvin-Benson-Bassham CCM Carbon-concentrating mechanism ClpP ATP-dependent Clp protease proteolytic subunit CO2 Carbon dioxide Col-0 Columbia-0 wild type Arabidopsis ecotype Cpn chaperonin

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