Rubisco Accumulation Factor 1 from T hermosynechococcus elongatus participates in the final stages of ribulose‐1,5‐bisphosphate carboxylase/oxygenase assembly in E scherichia coli cells and in vitro

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) biosynthesis is a multi‐step process in which specific chaperones are involved. Recently, a novel polypeptide, Rubisco Accumulation Factor 1 ( RAF 1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells ( Z ea mays ). However, neither its specific function nor its mode of action have as yet been determined. The results presented here show that the prokaryotic homolog of RAF 1 from T hermosynechococcus elongatus is expressed in cyanobacterial cells and interacts with a large Rubisco subunit (RbcL). Using a heterologous expression system, it was demonstrated that this protein promotes Rubisco assembly in E scherichia coli cells. Moreover, when co‐expressed with RbcL alone, a stable RbcL– RAF 1 complex is formed. Molecular mass determination for this Rubisco assembly intermediate by size‐exclusion chromatography coupled with multi‐angle light scattering indicates that it consists of an RbcL dimer and two RAF 1 molecules. A purified RbcL– RAF 1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL 8 ) core of Rubisco with RAF 1 results in disassembly of such a stucture and creation of an RbcL– RAF 1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process. Structured digital abstract   RAF1 physically interacts with RbcL by pull down ( View interaction )   RAF1 and RbcL bind by molecular sieving ( View interaction )