Dataset Information

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

ABSTRACT: 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 labelling (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, whereas 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Kristina Poljak

LAB HEAD: Markus Aebi

PROVIDER: PXD006435 | Pride | 2017-10-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20141219_01_p1579_elute.raw	Raw
	20141219_02_p1579_elute2.raw	Raw
	20141219_05_p1579_elute3.raw	Raw
	20161031_05_WTctrl_R2_30k.raw	Raw
	20161031_06_WTctrl_R2_60k.raw	Raw

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Quantitative Profiling of N-linked Glycosylation Machinery in Yeast <i>Saccharomyces cerevisiae</i>.

Poljak Kristina K Selevsek Nathalie N Ngwa Elsy E Grossmann Jonas J Losfeld Marie Estelle ME Aebi Markus M

Molecular & cellular proteomics : MCP 20171009 1

Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The <i>N</i>-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 a ...[more]

PMID: 28993419

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Project description:STT3A and STT3B are the main catalytic subunit of oligosaccharyltransferase (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and posttranslocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomics and glycoproteomics analyses to characterize the protein expression and glycosylation in STT3A-knockout (KO), STT3B-KO, and wild-type (WT) HEK293 cells. In total, 3,993 proteins, 4,020 unique N-linked intact glycopeptides (IGPs) and 723 glycosites, representing 401 glycoproteins were identified in KO and WT cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, mannosylated N-glycans from glycoproteins were reduced, while fucosylated N-glycans were increased in STT3A-KO cells. The glycan changes may be caused by the differential expression of glycosyltransferases and the activation of unfolded protein response (UPR) with further analysis of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells. We verified that the hyperglycosylation of ENPL was caused by the ER stress and UPR, which were induced by the deletion of the STT3A. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.STT3A and STT3B are the main catalytic subunit of oligosaccharyltransferase (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and posttranslocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomics and glycoproteomics analyses to characterize the protein expression and glycosylation in STT3A-knockout (KO), STT3B-KO, and wild-type (WT) HEK293 cells. In total, 3,993 proteins, 4,020 unique N-linked intact glycopeptides (IGPs) and 723 glycosites, representing 401 glycoproteins were identified in KO and WT cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, mannosylated N-glycans from glycoproteins were reduced, while fucosylated N-glycans were increased in STT3A-KO cells. The glycan changes may be caused by the differential expression of glycosyltransferases and the activation of unfolded protein response (UPR) with further analysis of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells. We verified that the hyperglycosylation of ENPL was caused by the ER stress and UPR, which were induced by the deletion of the STT3A. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.