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 ORCID: https://orcid.org/0000-0002-2576-7959, Lehrman, Mark A, Bütikofer, Peter and Menon, Anant K (2013) 'Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1.'. The Journal of Biological Chemistry, Vol 288, Issue 28, pp. 20616-20623.

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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.

Item Type:	Article
Subjects:	QU Biochemistry > Cells and Genetics > QU 350 Cellular structures QU Biochemistry > Genetics > QU 450 General Works QU Biochemistry > Proteins. Amino Acids. Peptides > QU 58.5 DNA. QX Parasitology > Protozoa > QX 70 Mastigophora. (e.g., Giardia. Trichomonas. Trypanosoma. Leishmania)
Faculty: Department:	Biological Sciences > Department of Tropical Disease Biology
Digital Object Identifer (DOI):	https://doi.org/10.1074/jbc.M113.479642
Depositing User:	Mary Creegan
Date Deposited:	06 Aug 2014 13:18
Last Modified:	27 Sep 2019 15:29
URI:	https://archive.lstmed.ac.uk/id/eprint/3821

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