Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is responsible for carbon dioxide conversion during photosynthesis and, therefore, is the most important protein in biomass generation. Modifications of this biocatalyst toward improvements in its properties are hindered by the complicated and not yet fully understood assembly process required for the formation of active holoenzymes. An entire set of auxiliary factors, including chaperonin GroEL/GroES and assembly chaperones RbcX or Rubisco accumulation factor 1 (RAF1), is involved in the folding and subsequent assembly of Rubisco subunits. Recently, it has been shown that cyanobacterial RAF1 acts during the formation of the large Rubisco subunit (RbcL) dimer. However, both its physiological function and its necessity in the prokaryotic Rubisco formation process remain elusive. Here, we demonstrate that the Synechocystis sp. PCC 6803 strain with raf1 gene disruption shows the same growth rate as wild-type cells under standard conditions. Moreover, the Rubisco biosynthesis process seems to be unperturbed in mutant cells despite the absence of RbcL-RAF1 complexes. However, in the tested environmental conditions, sulfur starvation triggers the degradation of RbcL and subsequent proteolysis of other polypeptides in wild-type but not Δraf1 strains. Pull-down experiments also indicate that, apart from Rubisco, RAF1 co-purifies with phycocyanins. We postulate that RAF1 is not an obligatory factor in cyanobacterial Rubisco assembly, but rather participates in environmentally regulated Rubisco homeostasis.
Highlights
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first reaction of the Calvin–Benson–Bassham cycle—incorporation of carbon dioxide into a sugar substrate, synthesizing two 3-phosphoglycerate molecules as a product
Disruption of the sll0102 gene encoding the Rubisco accumulation factor 1 (RAF1) homolog was performed via double crossover with transformation plasmid pTZ57raf1KO, in which ca. 2/3 of the raf1 sequence was replaced by a kanamycin-resistance cassette (Fig. 1a)
To verify whether RAF1 is a crucial factor in cyanobacterial Rubisco biosynthesis, we compared the growth rates of wild-type and Δraf1 cells as well as the amount of RbcL and quality of RbcL-containing complexes in crude extracts from these lines
Summary
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first reaction of the Calvin–Benson–Bassham cycle—incorporation of carbon dioxide into a sugar substrate, synthesizing two 3-phosphoglycerate molecules as a product. The establishment of a feasible and easy screening method for promising mutations is still hindered by the complicated and not fully understood Rubisco biosynthesis process, especially for the more complex form I of the enzyme. This form, which is met in eukaryotic photosynthetic organisms as well as in cyanobacteria, is built of eight large (RbcL) and eight small (RbcS) subunits, which have to be properly folded and assembled in a step-wise manner. Before that stage, a specific DnaJlike factor—Bsd, found in eukaryotic chloroplasts seems to be crucial during the folding of the N-terminal domain
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