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

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.