Heterologous expression and initial characterization of recombinant RbcX protein from Thermosynechococcus elongatus BP-1 and the role of RbcX in RuBisCO assembly.

Abstract

In the cyanobacterial RuBisCO operon from Thermosynechococcus elongatus the rbcX gene is juxtaposed and cotranscribed with the rbcL and rbcS genes which encode large and small RuBisCO subunits, respectively. It has been suggested that the rbcX position is not random and that the RbcX protein could be a chaperone for RuBisCO. In this study, the RbcX protein from T. elongatus was overexpressed, purified and preliminary functional studies were conducted. The recombinant protein purified from Escherichia coli extracts was predominantly present in a soluble fraction in a dimeric form. Coexpression experiments have demonstrated that RbcX can mediate RbcL dimer (L(2)) formation, and that it is essential for the L(8) core complex assembly. This is the first characterization of the RbcX protein from a thermophilic organism.

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

“…As it had been suggested above it is likely that RAF1 from T. elongatus interacts with RbcL, we decided to investigate whether this protein is able to support Rubisco assembly. To answer this question, we used a previously described approach to co‐express Rubisco‐encoding genes with genes of putative chaperones in E. coli cells . Bacteria transformed with rbcL , rbcS , rbcX or raf1 ‐carrying plasmids (see Experimental procedures for details) were grown and then subjected to protein extraction.…”

“…Elucidation of the function of Rubisco‐interacting chaperones by in vitro methods or using a heterologous expression system has been successfully performed previously . In contrast to eukaryotic photosynthetic proteins, their prokaryotic homologs are usually easily expressed in E. coli , which makes them suitable tools for studying the processes that are common to cyanobacterial and plant cells.…”

“…For the co‐expression experiment, a fragment containing the raf1 gene was amplified as described above but using primer raf1coFw instead of primer raf1Fw. After digestion with Bam HI endonuclease, the DNA fragment was cloned into pUC18rbcL and pUC18rbcLS plasmids , digested with the same restriction enzyme, and dephosphorylated using shrimp alkaline phosphatase (Fermentas). The resulting plasmid (pUC18LR) carried the T. elongatus rbcL gene followed by the raf1 gene oriented in the same direction.…”

Structure of the RuBisCO chaperone RbcX from the thermophilic cyanobacteriumThermosynechococcus elongatus

“…CA (Li & Tabita, 1997), Synechococcus sp. PCC 7002 (Onizuka et al, 2004) and Thermosynechococcus elongatus (Tarnawski, Gubernator et al, 2008). Thus, it seems that the presence of the rbcX gene within or outside the RuBisCO operon is closely related to its significance in RuBisCO assembly.…”

The state of oligomerization of Rubisco controls the rate of LSU translation inChlamydomonas reinhardtii

“…PCC7942 (Emlyn-Jones et al, 2006). Whether this dispensability still holds true for cyanobacterial species harboring a Rubisco LXS operon awaits further confirmation (Onizuka et al, 2004;Emlyn-Jones et al, 2006;Tarnawski et al, 2008). To date, there is no evidence of its requirement in algae and plants, where two RBCX isoforms, which would form homodimers, have been described (Kolesinski et al, 2013;Bracher et al, 2015).…”