title: De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes.
abstract: De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
authors.forename: Shaheen S
authors.initials: SS
authors.lastname: Sutterwala
authors.forename: Caleb H
authors.initials: CH
authors.lastname: Creswell
authors.forename: Sumana
authors.initials: S
authors.lastname: Sanyal
authors.forename: Anant K
authors.initials: AK
authors.lastname: Menon
authors.forename: James D
authors.initials: JD
authors.lastname: Bangs
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D058851:GPI-Linked Proteins; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D015800:Protozoan Proteins / Q000378:metabolism; D013107:Sphingolipids / Q000096:biosynthesis*; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D002518:Ceramides; D005229:Fatty Acids, Monounsaturated; C102519:GML protein, human; D058851:GPI-Linked Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D013107:Sphingolipids; D014643:Variant Surface Glycoproteins, Trypanosoma; C001996:thermozymocidin