Glycosylphosphatidylinositol-dependent protein trafficking in bloodstream stage Trypanosoma brucei.
We have previously demonstrated that glycosylphosphatidylinositol (GPI) anchors strongly influence protein trafficking in the procyclic insect stage of Trypanosoma brucei (M. A. McDowell, D. A. Ransom, and J. D. Bangs, Biochem. J. 335:681-689, 1998), where GPI-minus variant surface glycoprotein (VSG) reporters have greatly reduced rates of endoplasmic reticulum (ER) exit but are ultimately secreted. We now demonstrate that GPI-dependent trafficking also occurs in pathogenic bloodstream trypanosomes. However, unlike in procyclic trypanosomes, truncated VSGs lacking C-terminal GPI-addition signals are not secreted but are mistargeted to the lysosome and degraded. Failure to export these reporters is not due to a deficiency in secretion of these cells since the N-terminal ATPase domain of the endogenous ER protein BiP is efficiently secreted from transgenic cell lines. Velocity sedimentation experiments indicate that GPI-minus VSG dimerizes similarly to wild-type VSG, suggesting that degradation is not due to ER quality control mechanisms. However, GPI-minus VSGs are fully protected from degradation by the cysteine protease inhibitor FMK024, a potent inhibitor of the major lysosomal protease trypanopain. Immunofluorescence of cells incubated with FMK024 demonstrates that GPI-minus VSG colocalizes with p67, a lysosomal marker. These data suggest that in the absence of a GPI anchor, VSG is mistargeted to the lysosome and subsequently degraded. Our findings indicate that GPI-dependent transport is a general feature of secretory trafficking in both stages of the life cycle. A working model is proposed in which GPI valence regulates progression in the secretory pathway of bloodstream stage trypanosomes.
Triggs
Veronica P
VP
Department of Biomolecular Chemistry, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA.
Bangs
James D
JD
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D001769:Blood / Q000469:parasitology*; D012634:Bodily Secretions / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000172:deficiency* / Q000235:genetics; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology*; D014346:Trypanosoma brucei brucei / Q000378:metabolism* / Q000472:pathogenicity; D014353:Trypanosomiasis, African / Q000097:blood* / Q000503:physiopathology; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D017261:Glycosylphosphatidylinositols; D014643:Variant Surface Glycoproteins, Trypanosoma; C106539:p67 deglycosylase; D000118:Acetylglucosaminidase; D003546:Cysteine Endopeptidases; C100684:trypanopain Tb