Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein.
N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.
Helenius
Jonne
J
Institute of Microbiology, Swiss Federal Institute of Technology, Zürich, CH-8092 Zürich, Switzerland.
Ng
Davis T W
DT
Marolda
Cristina L
CL
Walter
Peter
P
Valvano
Miguel A
MA
Aebi
Markus
M
D001216:Asparagine / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D017124:Conserved Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D007425:Intracellular Membranes / Q000378:metabolism; D050356:Lipid Metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D009844:Oligosaccharides / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009844:Oligosaccharides; D049294:Phospholipid Transfer Proteins; D011104:Polyisoprenyl Phosphate Oligosaccharides; C486728:RFT1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C058641:mannosyl(5)-N-acetyl(2)-glucose diphosphate dolichol; D001216:Asparagine; C058642:mannosyl(5)-N-acetyl(2)-glucose