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

• Published in: The FEBS journal Vol. 281; no. 17; pp. 3920 - 3932
• Main Authors: Kolesinski, Piotr, Belusiak, Iwona, Czarnocki‐Cieciura, Mariusz, Szczepaniak, Andrzej
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-09-2014
• Subjects: Bacterial Proteins - metabolism; Biosynthesis; chaperone proteins; Cyanobacteria - metabolism; E coli; Escherichia coli; Escherichia coli - enzymology; Microbiology; Molecular biology; Molecular Chaperones - metabolism; photosynthesis; Polypeptides; protein assembly; Proteins; RAF1; Ribulose-Bisphosphate Carboxylase - biosynthesis; Ribulose-Bisphosphate Carboxylase - metabolism; Rubisco biosynthesis; Thermosynechococcus elongatus; Zea mays; chaperone proteins; RAF1; photosynthesis; protein assembly; Rubisco biosynthesis
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12928

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.
Structured digital
 RAF1 physically interacts with RbcL by pull down (View interaction)
 RAF1 and RbcL bind by molecular sieving (View interaction)
Rubisco biosynthesis is a process in which specific chaperones (GroEL/ES, RbcX, RAF1) are involved. Cyanobacterial homolog of RAF1 protein interacts with RbcL and promotes Rubisco assembly in Escherichia coli cells. The addition of RbcS to RbcL2‐RAF12 complex leads to the active holoenzyme formation. Titration of the Rubisco core (RbcL8) with the RAF1 results in the creation of an RbcL2‐RAF12 intermediate.