Quantitative Profiling of N-linked Glycosylation Machinery in Yeast Saccharomyces cerevisiae .

Autor:	Poljak K; From the ‡Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland., Selevsek N; §Functional Genomics Center Zurich, UZH/ETH Zurich, CH-8057 Zurich, Switzerland., Ngwa E; From the ‡Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland., Grossmann J; §Functional Genomics Center Zurich, UZH/ETH Zurich, CH-8057 Zurich, Switzerland., Losfeld ME; From the ‡Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland., Aebi M; From the ‡Institute of Microbiology, ETH Zurich, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland; markus.aebi@micro.biol.ethz.ch.
Jazyk:	angličtina
Zdroj:	Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2018 Jan; Vol. 17 (1), pp. 18-30. Date of Electronic Publication: 2017 Oct 09.
DOI:	10.1074/mcp.RA117.000096
Abstrakt:	Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N -linked glycosylation machinery involves enzymes responsible for the assembly of the lipid-linked oligosaccharide (LLO), which is then transferred to the asparagine residues on the polypeptides by the enzyme oligosaccharyltransferase (OST). A major goal in the study of protein glycosylation is to establish quantitative methods for the analysis of site-specific extent of glycosylation. We developed a sensitive approach to examine glycosylation site occupancy in Saccharomyces cerevisiae by coupling stable isotope labeling (SILAC) approach to parallel reaction monitoring (PRM) mass spectrometry (MS). We combined the method with genetic tools and validated the approach with the identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of OST. Based on the observations that alternations in LLO substrate structure and OST subunits activity differentially alter the systemic output of OST, we conclude that sequon recognition is a direct property of the catalytic subunit Stt3p, auxiliary subunits such as Ost3p and Ost6p extend the OST substrate range by modulating interfering pathways such as protein folding. In addition, our proteomics approach revealed a novel regulatory network that connects isoprenoid lipid biosynthesis and LLO substrate assembly. (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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