Evolutionary divergence and functional insights into the heteromeric cis-prenyltransferase of Paramecium tetraurelia.

The biosynthesis of polyprenyl/dolichyl phosphate, an essential lipid carrier in protein glycosylation, occurs across all domains of life. Eukaryotic heteromeric enzymes involved in polyprenyl chain elongation consist of a highly conserved catalytic cis-prenyltransferase subunit (CPT-CS) and a less conserved CPT-accessory subunit (CPT-AS). Here, we present the first experimental evidence that dolichol biosynthesis in Paramecium tetraurelia is mediated by a heteromeric CPT complex. Using a multidisciplinary experimental approach, we identified two highly homologous catalytic CPT subunits, CPT1a and CPT1b, which exhibit high sequence similarity to other eukaryotic CPTs, along with a unique CPT-AS, named POC1 (partner of CPT1), which is a structural and functional relative of the human dehydrodolichyl diphosphate synthase complex subunit NUS1 (also known as NgBR) and yeast Nus1 CPT-AS. Despite low sequence similarity to other CPT-ASs, it retained a well-preserved C-terminal substrate-binding domain characteristic of its eukaryotic and prokaryotic counterparts. The loss of POC1 or CPT1a, but not CPT1b, results in a deficit in dolichol production, leading to a significant reduction in glycoprotein content and, ultimately, to the P. tetraurelia cell death. In a heterologous yeast system, both CPTs in complex with POC1 synthesized polyprenyl chains. The identification of a POC1 protein so distinct from other CPT-ASs may spark further efforts to uncover CPT-AS proteins in pathogenic protozoa, which have so far eluded detection despite phylogenetic evidences that CPT of Apicomplexa and Trichomonas sp. are heteromeric enzymes. Given their substantial sequence divergence from human NgBR and its animal orthologues, these protozoan CPT-ASs could represent highly specific targets for antiparasitic therapies.