Deletion of the GPIdeAc gene alters the location and fate of glycosylphosphatidylinositol precursors in Trypanosoma brucei.
Glycosylphosphatidylinositol (GPI) membrane anchors are ubiquitous among the eukaryotes. In most organisms, the pathway of GPI biosynthesis involves inositol acylation and inositol deacylation as discrete steps at the beginning and end of the pathway, respectively. The bloodstream form of the protozoan parasite Trypanosoma brucei is unusual in that these reactions occur on multiple GPI intermediates and that it can express side chains of up to six galactose residues on its mature GPI anchors. An inositol deacylase gene, T. brucei GPIdeAc, has been identified. A null mutant was created and shown to be capable of expressing normal mature GPI anchors on its variant surface glycoprotein. Here, we show that the null mutant synthesizes galactosylated forms of the mature GPI precursor, glycolipid A, at an accelerated rate (2.8-fold compared to wild type). These free GPIs accumulate at the cell surface as metabolic end products. Using continuous and pulse-chase labeling experiments, we show that there are two pools of glycolipid A. Only one pool is competent for transfer to nascent variant surface glycoprotein and represents 38% of glycolipid A in wild-type cells. This pool rises to 75% of glycolipid A in the GPIdeAc null mutant. We present a model for the pathway of GPI biosynthesis in T. brucei that helps to explain the complex phenotype of the GPIdeAc null mutant.
Güther
M Lucia S
ML
Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, The School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Prescott
Alan R
AR
Ferguson
Michael A J
MA
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D017353:Gene Deletion; D017125:Genes, Protozoan; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D013347:Subcellular Fractions / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism
D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D011498:Protein Precursors; D015800:Protozoan Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C436709:GPIdeAc protein, Trypanosoma brucei; D010744:Phosphoric Monoester Hydrolases; D005690:Galactose