The suppression of galactose metabolism in procylic form Trypanosoma brucei causes cessation of cell growth and alters procyclin glycoprotein structure and copy number.
Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4'-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDP-Glc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins., were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.
Roper
Janine R
JR
Division of Biological Chemistry and Molecular Microbiology, The School of Life Sciences, University of Dundee, Scotland, UK.
Güther
M Lucia S
ML
Macrae
James I
JI
Prescott
Alan R
AR
Hallyburton
Irene
I
Acosta-Serrano
Alvaro
A
Ferguson
Michael A J
MA
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D019281:Dimerization; D005690:Galactose / Q000378:metabolism*; D017353:Gene Deletion; D017125:Genes, Protozoan; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008830:Microbodies / Q000201:enzymology; D015394:Molecular Structure; D010641:Phenotype; D020836:Protein Structure, Quaternary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; C053277:procyclic acidic repetitive protein, Trypanosoma; D014534:UDPglucose 4-Epimerase; D005690:Galactose