Two Plasmodium falciparum Ribonucleotide Reductase Small Subunits, PfR2 and PfR4, Interact with Each Other and are Components of the in vivo Enzyme Complex Original Research Article

Ribonucleotide reductase (RNR) is a tetrameric enzyme, composed of two large (R1) and two small (R2) subunits, which regulates the nucleotide balance in cells by controlling the rate-limiting step for deoxyribonucleotide synthesis. We have identified a second copy of the small subunit gene, termed PfR4, encoding a 324 amino acid residue polypeptide that shares only 25% identity with the previously identified PfR2 small subunit of Plasmodium falciparum. PfR4 expression is cell-cycle-regulated, and the profile of transcript and protein expression corresponds to that of PfR2. A 1.3 kb PfR4 5′-flanking fragment contained a functional promoter activity. We have detected interaction between PfR2 and PfR4 by co-immunoprecipitation experiments. Indirect immunofluorescence analysis showed distinct localization of two small RNR subunits with some colocalization. The association of PfR1 large subunit with PfR4 was detected by GST pull-down assay. This interaction is reduced significantly when using a PfR4 truncated at the COOH terminus, suggesting the involvement of COOH-terminal residues in PfR4–PfR1 interaction. All three RNR subunits co-eluted on a Superose 12 size-exclusion column corresponding to fractions with a molecular mass of around 250 kDa. This suggests the existence of all three RNR subunits in Plasmodium in a native complex of α2ββ′ configuration.