title: Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana.
abstract: The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria 3010, Australia.
authors.forename: K A
authors.initials: KA
authors.lastname: Mullin
authors.forename: B J
authors.initials: BJ
authors.lastname: Foth
authors.forename: S C
authors.initials: SC
authors.lastname: Ilgoutz
authors.forename: J M
authors.initials: JM
authors.lastname: Callaghan
authors.forename: J L
authors.initials: JL
authors.lastname: Zawadzki
authors.forename: G I
authors.initials: GI
authors.lastname: McFadden
authors.forename: M J
authors.initials: MJ
authors.lastname: McConville
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D004396:Coloring Agents / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D007894:Leishmania mexicana / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure*; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D017261:Glycosylphosphatidylinositols; D011993:Recombinant Fusion Proteins; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase