Dual Functions of a Rubisco Activase in Metabolic Repair and Recruitment to Carboxysomes.

Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.

Role of auxiliary proteins in Rubisco biogenesis and function.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the conversion of atmospheric CO2 into organic compounds during photosynthesis. Despite its pivotal role in plant metabolism, Rubisco is an inefficient enzyme and has therefore been a key target in bioengineering efforts to improve crop yields. Much has been learnt about the complex cellular machinery involved in Rubisco assembly and metabolic repair over recent years. The simple form of Rubisco found in certain bacteria and dinoflagellates comprises two large subunits, and generally requires the chaperonin system for folding. However, the evolution of hexadecameric Rubisco, which comprises eight large and eight small subunits, from its dimeric precursor has rendered Rubisco in most plants, algae, cyanobacteria and proteobacteria dependent on an array of additional factors. These auxiliary factors include several chaperones for assembly as well as ATPases of the AAA+ family for functional maintenance. An integrated view of the pathways underlying Rubisco biogenesis and repair will pave the way for efforts to improve the enzyme with the goal of increasing crop yields.

A beta-herpesvirus with fluorescent capsids to study transport in living cells.

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion.