title: O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins.
abstract: Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA.
authors.forename: Quira
authors.initials: Q
authors.lastname: Zeidan
authors.forename: Zihao
authors.initials: Z
authors.lastname: Wang
authors.forename: Antonio
authors.initials: A
authors.lastname: De Maio
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Hart
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D051381:Rats; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000737:chemistry; D054683:Ribosome Subunits, Large, Eukaryotic / Q000378:metabolism; D012270:Ribosomes / Q000201:enzymology
chemical_list: D012269:Ribosomal Proteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine
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