Functional analysis of the plastid and nuclear encoded CbbX proteins of Cyanidioschyzon merolae

Abstract

CbbX is believed to be a transcriptional regulator of the subunit genes (rbcL and rbcS) of RuBisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase) as well as possibly a molecular chaperon of RuBisCO subunit assembly. The unicellular red alga Cyanidioschyzon merolae strain 10D possesses two distinct cbbX genes; one is part of the plastid genome and the other is found in the cell nucleus, whereas the RuBisCO operon (rbcL-rbcS-cbbX) is located only on the plastid genome. We examined the role of CbbX proteins of C. merolae in the expression of the RuBisCO operon. First, His-tagged nuclear and plastid CbbX proteins were produced in Escherichia coli and purified by affinity column chromatography. Both proteins showed binding activity to upstream of the coding region of rbcL. Yeast two hybrid analysis showed direct interaction between nuclear and plastid CbbX proteins but no interaction were found among CbbX, RbcL and RbcS. Then the transcription initiation site of the RuBisCO operon of C. merolae was determined. Next, in order to examine the role of CbbX in vivo, we constructed a plasmid carrying the promoter region of the RuBisCO operon fused to Escherichia coli lacZ, and introduced it into E. coli cells into which a cloned nuclear or plastid cbbX gene under IPTG inducible promoter control was also introduced. Expression of LacZ in the transformed E.coli was observed. Enforced expression of either one of the cbbX genes resulted in a remarkable reduction of lacZ expression suggesting that CbbXs are rather transcriptional regulators than the molecular chaperon of RuBisCO. We discuss the mechanism by which the nuclear and plastid CbbX proteins regulate the RuBisCO operon of C. merolae.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

“…Similar to C . tobin , copies of cbbX were found in earlier studies of the cryptophyte Guillardia theta [ 84 ], where cbbX copies were found in the nucleomorph as well as chloroplast of this alga, followed by observations in the rhodophyte Cyanidioschyzon merolae [ 86 ] where cbbX was seen to occur in the nucleus as well as the chloroplast. Below we expand on preliminary observations that suggested that a broad phylogenetic occurrence of a chloroplast/nuclear cbb X coding duality exists in red lineage algae [ 87 ].…”

Characterization of the heterooligomeric red-type rubisco activase from red algae

“…S1). These isoforms have been produced in E. coli previously, but were not tested for activase function (30). Following overexpression in E. coli, CmN was easily obtained at high purity by using a combination of affinity, anion-exchange, and size-exclusion chromatography ( Fig.…”

Nucleus-Independent Control of the Rubisco Operon by the Plastid-Encoded Transcription Factor Ycf30 in the Red Alga Cyanidioschyzon merolae

“…1A). Using primer extension mapping, a unique transcription initiation site was identified under dark and light conditions (Fujita et al, 2008b). Thus, transcription from the rbcL promoter was increased in response to the light.…”

“…3A) mapped the binding site to the region from -59 to -30 (Fig. 3C), although the addition of 30-bp oligonucleotides seemed to cause a variation in the (Fujita et al, 2008b), translation initiation codon, and putative -10 box are indicated. Based on LTTR recognition sequence, three sequences (a-c) in the region from -59 to -30 are shown below the sequence.…”