Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco
peer-reviewed · Journal of Biological Chemistry · 2020
| Date | 2020-11-01 |
| Type | peer-reviewed |
| Venue | Journal of Biological Chemistry |
| Publisher | American Society for Biochemistry and Molecular Biology |
| Contribution | downstream-application |
| DOI | 10.1074/jbc.RA120.015759 |
Abstract
The photosynthetic CO 2 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. Toward 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 in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.
Methods and tools
- PEAKS: Commercial DP-based de novo
- Rubisco large subunit N-terminal determination: De novo sequencing used to establish what the N terminus of a recombinant protein actually is, which the biological argument then rests on. Arabidopsis Rubisco large subunit variants expressed in E. coli are processed unpredictably by the host, so the construct sequence does not tell you which residues survive. Purified proteins were run on SDS gels, the RbcL band excised, and the N termini determined by de novo mass spectrometric sequencing in PEAKS Studio X+, alongside a semi-specific tryptic database search and peak-area quantification; the work was done as a service by the Bioprocessing Technology Institute, A*STAR. The result is Table 1 in full: the observed N termini for wild type and each truncation variant with their peak-area ratios, showing for instance that wild type is a mixture of MSPQTETKAS, SPQTETKAS and PQTETKAS at 36, 15 and 49 percent. Those assignments are what let the paper claim that large subunits lacking residues 1 to 4 are functional carboxylases that Rubisco activase cannot activate, and they drive a further inference about cooperativity, since some deltaN2 N termini proved identical to deltaN3 ones. A tryptic database search alone would not have found them, because host processing leaves ragged non-tryptic N termini.