The trypanosome UDP-glucose pyrophosphorylase is imported by piggybacking into glycosomes where unconventional sugar nucleotide synthesis takes place

Abstract

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways usually present in the cytosol of other eukaryotes, such as glycolysis and biosynthesis of sugar nucleotides. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localised in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisomal targeting signal (PTS1 and PTS2). The questions we addressed here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotide functional and (ii) how the PTS-free UGP is imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3-10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knock-down UGP mutant cell line (RNAiUGP) was rescued by expressing a recoded UGP in the organelle (RNAiUGP/EXPrUGP-GPDH). Our conclusion was supported by targeted metabolomic analyses (IC-HRMS) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. Importance Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localisation of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here we showed that the cytosolic and glycosomal pathways are functional. Like in all other eukaryotes, the cytosolic pathways feed glycosylation reactions, however the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK), which are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.