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Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1
Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endo...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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American Society for Biochemistry and Molecular Biology
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711325/ https://www.ncbi.nlm.nih.gov/pubmed/23720757 http://dx.doi.org/10.1074/jbc.M113.479642 |
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author | Jelk, Jennifer Gao, Ningguo Serricchio, Mauro Signorell, Aita Schmidt, Remo S. Bangs, James D. Acosta-Serrano, Alvaro Lehrman, Mark A. Bütikofer, Peter Menon, Anant K. |
author_facet | Jelk, Jennifer Gao, Ningguo Serricchio, Mauro Signorell, Aita Schmidt, Remo S. Bangs, James D. Acosta-Serrano, Alvaro Lehrman, Mark A. Bütikofer, Peter Menon, Anant K. |
author_sort | Jelk, Jennifer |
collection | PubMed |
description | Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man(5)GlcNAc(2)-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. |
format | Online Article Text |
id | pubmed-3711325 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-37113252013-07-19 Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 Jelk, Jennifer Gao, Ningguo Serricchio, Mauro Signorell, Aita Schmidt, Remo S. Bangs, James D. Acosta-Serrano, Alvaro Lehrman, Mark A. Bütikofer, Peter Menon, Anant K. J Biol Chem Glycobiology and Extracellular Matrices Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man(5)GlcNAc(2)-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. American Society for Biochemistry and Molecular Biology 2013-07-12 2013-05-28 /pmc/articles/PMC3711325/ /pubmed/23720757 http://dx.doi.org/10.1074/jbc.M113.479642 Text en © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/) applies to Author Choice Articles |
spellingShingle | Glycobiology and Extracellular Matrices Jelk, Jennifer Gao, Ningguo Serricchio, Mauro Signorell, Aita Schmidt, Remo S. Bangs, James D. Acosta-Serrano, Alvaro Lehrman, Mark A. Bütikofer, Peter Menon, Anant K. Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 |
title | Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 |
title_full | Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 |
title_fullStr | Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 |
title_full_unstemmed | Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 |
title_short | Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1 |
title_sort | glycoprotein biosynthesis in a eukaryote lacking the membrane protein rft1 |
topic | Glycobiology and Extracellular Matrices |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711325/ https://www.ncbi.nlm.nih.gov/pubmed/23720757 http://dx.doi.org/10.1074/jbc.M113.479642 |
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