title: Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1.
abstract: Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.
authors.affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jelk
authors.forename: Ningguo
authors.initials: N
authors.lastname: Gao
authors.forename: Mauro
authors.initials: M
authors.lastname: Serricchio
authors.forename: Aita
authors.initials: A
authors.lastname: Signorell
authors.forename: Remo S
authors.initials: RS
authors.lastname: Schmidt
authors.forename: James D
authors.initials: JD
authors.lastname: Bangs
authors.forename: Alvaro
authors.initials: A
authors.lastname: Acosta-Serrano
authors.forename: Mark A
authors.initials: MA
authors.lastname: Lehrman
authors.forename: Peter
authors.initials: P
authors.lastname: Bütikofer
authors.forename: Anant K
authors.initials: AK
authors.lastname: Menon
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005947:Glucose / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011134:Polysaccharides; D015800:Protozoan Proteins; C486728:RFT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C058641:mannosyl(5)-N-acetyl(2)-glucose diphosphate dolichol; D005947:Glucose
keywords: Endoplasmic Reticulum (ER); Flippase; Glycobiology; Glycoprotein Biosynthesis; Lipid Synthesis; Membrane Proteins; Trypanosoma brucei