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Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

ABSTRACT: In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michele Tinti

LAB HEAD: Mike Ferguson

PROVIDER: PXD023124 | Pride | 2021-01-28

REPOSITORIES: Pride

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			Action	DRS
	LG-1hr-B10.mgf	Mgf
	LG1-100mM.raw	Raw
	LG1-150mM.raw	Raw
	LG1-200mM.raw	Raw
	LG1-20mM.raw	Raw

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The trypanosome UDP-glucose pyrophosphorylase is active in both the glycosomal and cytosolic compartments and is imported into glycosomes by piggybacking

Project description:Trypanosoma brucei, the etiological agent of sleeping sickness, has particular peroxisome-like organelles called glycosomes where several metabolic pathways are compartmentalized, including glycolysis. Many enzymes from sugar nucleotide biosynthetic pathways are located in this organelle, contrasting with the cytosolic localisation reported in the eukaryota domain. The UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for UDP-glucose (UDP-Glc) production, is essential for growth and survival of trypanosomes. In addition, UGP is localized in both glycosomes and cytosol, raising questions about why would these parasites maintain a pathway functional in two different subcellular compartments. UGP is imported into glycosomes by piggybacking on the glycosomal protein PEPCK, which is the first evidence of piggybacking involving two proteins not functionally related, and the first report in trypanosomatids. The dataset demonstrates that UGP is present in the total cellular extract and the glycocomal fraction of the wild-type trypanosomes, while only in the total cellular extract of the PEPCK null mutant.

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A high-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC

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Proteomic identification of the UDP-GlcNAc : PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

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