title: Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro.
abstract: 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 (RAF1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells (Zea 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 RAF1 from Thermosynechococcus 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 Escherichia coli cells. Moreover, when co-expressed with RbcL alone, a stable RbcL-RAF1 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 RAF1 molecules. A purified RbcL-RAF1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL8) core of Rubisco with RAF1 results in disassembly of such a stucture and creation of an RbcL-RAF1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process.
authors.affiliation: Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Kolesinski
authors.forename: Iwona
authors.initials: I
authors.lastname: Belusiak
authors.forename: Mariusz
authors.initials: M
authors.lastname: Czarnocki-Cieciura
authors.forename: Andrzej
authors.initials: A
authors.lastname: Szczepaniak
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D000458:Cyanobacteria / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D018832:Molecular Chaperones / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase
keywords: RAF1; Rubisco biosynthesis; chaperone proteins; photosynthesis; protein assembly