Chaperones for folding, assembly and maintenance of Rubisco

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for the fixation of atmospheric CO2 in photosynthesis. Rubisco is not only the most abundant protein in nature but also one with a remarkably high chaperone requirement for folding, assembly and maintenance. The major form of Rubisco (form I) is hexadecameric, consisting of 8 large (RbcL) and 8 small (RbcS) subunits. The form I Rubiscos are phylogenetically divided into a green branch, present in cyanobacteria, green algae and plants, and a red branch, present mainly in photosynthetic bacteria, red algae and phytoplankton. The red-type Rubiscos are responsible for most oceanic CO2 uptake and many are able to better distinguish between CO2 and O2 than their plant counterparts. Recently, we showed that RbcL subunit folding by the GroEL/ES chaperonin is tightly coupled with assembly mediated by the assembly chaperone RbcX. Specifically, RbcX captures the flexible C-terminal segment of one RbcL subunit in its central binding cleft and binds another subunit via its peripheral binding region. Upon binding of RbcS subunits, the RbcL8RbcX8 complex undergoes an allosteric structural change that results in the dissociation of RbcX and formation of stable Rubisco holoenzyme. Before the assembled Rubisco can proceed with photosynthesis, its enzymatic center has to be activated. To acquire and maintain catalytic activity, Rubisco must first be carbamylated by a non-substrate CO2 molecule at the active-site lysine and then bind a Mg2+ ion as cofactor. Premature binding of the substrate ribulose 1,5-biphosphate (RuBP) to uncarbamylated Rubisco results in an inactive complex. Reactivation is catalyzed by a AAA+ (ATPases associated with various cellular activities) protein called Rubisco activase (Rca), which releases the inhibitory RuBP in an ATP-dependent manner. Recently, we solved the crystal structures of green plant Rca from tobacco and the red-type Rca from the proteobacterium Rhodobacter sphaeroides. Negative stain EM and biochemical analysis showed that ATP and RuBP must bind to convert the Rhodobacter activase, called CbbX, into functionally active, hexameric rings. Tobacco Rca, on the other hand, forms functional hexamers in the absence of ATP and has no binding site for RuBP. Furthermore, the CbbX ATPase is strongly stimulated upon interaction with Rubisco, while Tobacco Rca is constitutively ATPase active. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the narrow hexamer pore, while Tobacco Session 7. Protein Transport and Assembly