Localization and phylogenetic analysis of enzymes related to organellar genome replication in the unicellular rhodophyte Cyanidioschyzon merolae

Abstract

Plants and algae possess plastids and mitochondria harboring their own genomes, which are replicated by the apparatus consisting of DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, and primer removal enzyme. In the higher plant Arabidopsis thaliana, organellar replication-related enzymes (OREs) are similar in plastids and mitochondria because many of them are dually targeted to plastids and mitochondria. In the red algae, there is a report about a DNA replicase, plant/protist organellar DNA polymerase, which is localized to both plastids and mitochondria. However, other OREs remain unclear in algae. Here, we identified OREs possibly localized to organelles in the unicellular rhodophyte Cyanidioschyzon merolae. We then examined intracellular localization of green fluorescent protein-fusion proteins of these enzymes in C. merolae, whose cell has a single plastid and a single mitochondrion and is suitable for localization analysis, demonstrating that the plastid and the mitochondrion contain markedly different components of replication machinery. Phylogenetic analyses revealed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteobacterial, cyanobacterial, and eukaryotic, in both red algae and green plants. Especially in the red alga, many enzymes of cyanobacterial origin remained. Finally, on the basis of the results of localization and phylogenetic analyses, we propose a model on the succession of OREs in eukaryotes.

Keywords: GFP-fusion proteins; evolution of replication apparatus; mitochondria and plastids; red algae; subcellular localization.

Fig. 1.—

Localization of ORE-GFP fusion proteins in C. merolae cell. (A) Fluorescence images with DAPI staining of an interphase cell and a dividing cell. Nuc; Nucleus, Mt; Mitochondrion, Pt; Plastid. (B) Schematic representation of the constructs expressing GFP fusion proteins under the control of the apcC promoter of C. merolae (PapcC) and the NOS terminator (nos). Inserts corresponding to transit peptide regions (TP) were cloned into the pCG1 vector. (C–E) Fluorescence microscopic images of the transiently transformed C. merolae cells. Images of OREs localized to plastid (C), mitochondrion (D), or both plastid and mitochondrion (E) are shown.

Fig. 2.—

Simplified phylogenetic tree of OREs originating from bacteria. In rhodophytes, enzymes of cyanobacterial origin are DnaB, DnaG (A), gyrases (B), and TOP1 (C). Enzymes of α-proteobacterial origin are SSB and Pol I (D). Detailed phylogenetic trees are shown in supplementary figure S4, Supplementary Material online.

Fig. 3.—

Schematic illustration of the processes leading to succession of OREs in eukaryotes. Boxes consisting of six small boxes indicate component enzymes of organellar replication, and explanatory notes for each location of boxes are shown at the lower left corner of this figure. Organelle names, mitochondria (Mt), and plastid (Pt), are indicated in the upper left corner of each box. The enzymes that originated from α-proteobacteria and cyanobacteria are shown in orange and blue boxes, respectively. Eukaryotic enzymes, whose origin is unclear, are shown in pink boxes. Polγ is conserved only in opisthokonts including animals and fungi, and the enzyme is shown in a purple box. Bacterial enzymes derived from unknown origin are shown in gray boxes. A land-plant-specific enzyme, OSB, is shown in green boxes. ssDNA binding protein in plastids of rhodophytes was not identified in the present study, and the box is indicated by a question mark.