title: Glycosylphosphatidylinositol-dependent secretory transport in Trypanosoma brucei.
abstract: We have investigated the role of glycosylphosphatidylinositol (GPI) anchors in forward secretory trafficking using African trypanosomes as a model system. Soluble GPI-minus forms of variant surface glycoprotein (VSG), in which the C-terminal GPI-addition peptide signal is deleted, are secreted from transformed procyclic trypanosomes with 5-fold reduced kinetics, relative to matched GPI-anchored constructs. Cell fractionation and immunofluorescence localization studies indicate that the GPI-minus VSG reporters accumulate in the endoplasmic reticulum (ER). This transport defect is specific, since overexpression of GPI-minus VSG has no effect on the rate of transport of a second soluble secretory reporter (BiPN) when co-expressed in the same cells. Two results suggest that delayed forward transport cannot be accounted for by failure to fold/assemble in the absence of a GPI anchor, thereby leading to prolonged association with ER quality-control machinery. First, no evidence was found for elevated association of GPI-minus VSG with the ER molecular chaperone, BiP. Secondly, newly synthesized GPI-minus VSG is dimerized efficiently, as judged by velocity-sedimentation analysis. GPI-dependent transport is not confined to the VSG reporters, because a similar dependence is found with another trypanosomal GPI-anchored protein, trans-sialidase. These findings suggest that GPI structures act in a positive manner to mediate efficient forward transport of some, and perhaps all, GPI-anchored proteins in the early secretory pathway of trypanosomes. Possible mechanisms for GPI-dependent transport are discussed with respect to current models of vesicular trafficking.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI 53706, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: McDowell
authors.forename: D M
authors.initials: DM
authors.lastname: Ransom
authors.forename: J D
authors.initials: JD
authors.lastname: Bangs
mesh_terms: D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D009439:Neuraminidase / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000502:physiology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D009439:Neuraminidase