O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins.
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.
Zeidan
Quira
Q
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA.
Wang
Zihao
Z
De Maio
Antonio
A
Hart
Gerald W
GW
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
D012269:Ribosomal Proteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine
Mol Cell Proteomics. 2007 Aug;6(8):1365-79
17507370
Cell. 2005 Sep 23;122(6):957-68
16169070
Nat Biotechnol. 2005 Jan;23(1):94-101
15592455
Biochem Soc Trans. 2006 Apr;34(Pt 2):213-6
16545079
Gene. 2007 Oct 15;401(1-2):1-3
17689889
Nature. 2005 Jan 6;433(7021):77-83
15635413
Proc Natl Acad Sci U S A. 1988 May;85(10):3324-8
3368443
J Biol Chem. 2001 Mar 30;276(13):9838-45
11148210
EMBO J. 2001 Jun 1;20(11):2867-74
11387219
Biochimie. 2001 Oct;83(10):919-31
11728629
J Biol Chem. 1989 Dec 5;264(34):20620-4
2511207
Nat Rev Mol Cell Biol. 2004 Oct;5(10):827-35
15459663
J Biol Chem. 2005 Sep 23;280(38):32944-56
16027160
Nat Rev Mol Cell Biol. 2007 Jul;8(7):574-85
17519961
Structure. 2008 Apr;16(4):535-48
18400176
Nat Cell Biol. 2008 Oct;10(10):1224-31
18794846
AIDS Res Hum Retroviruses. 1993 Feb;9(2):115-21
8457378
J Biol Chem. 1988 Aug 15;263(23):11473-7
3403539
Biochem Cell Biol. 1995 Nov-Dec;73(11-12):933-47
8722009
J Biochem. 1967 Jun;61(6):778-86
6061691
Cell. 2007 Dec 14;131(6):1190-203
18083107
Nature. 2000 Sep 21;407(6802):327-39
11014182
J Biol Chem. 1994 Jul 29;269(30):19321-30
8034696
Cell. 1993 Feb 12;72(3):443-57
8431947
Mol Microbiol. 2006 Mar;59(6):1651-63
16553873
J Biol Chem. 2005 Jan 14;280(2):1360-8
15516338
J Biol Chem. 2004 Jul 16;279(29):30133-42
15138254
Arch Biochem Biophys. 1999 Feb 1;362(1):38-45
9917327
J Proteome Res. 2007 Nov;6(11):4150-62
17924679
Cell. 2001 Nov 30;107(5):679-88
11733066
Mol Cell Biol. 2004 Apr;24(8):3112-24
15060135
Biochim Biophys Acta. 2002 Apr 12;1574(3):255-61
11997090
Nat Chem Biol. 2007 Jun;3(6):339-48
17496889
Cell. 2000 Jul 7;102(1):67-76
10929714
Cell. 2003 Oct 17;115(2):187-98
14567916
Anal Biochem. 2001 Jun 15;293(2):169-77
11399029
Am J Physiol Endocrinol Metab. 2008 Jul;295(1):E17-28
18445751
J Cell Biochem. 1998 Jun 1;69(3):244-51
9581863
Nat Med. 1997 Dec;3(12):1383-8
9396609
Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):539-43
1346232
Biochem J. 2007 Apr 15;403(2):217-34
17376031
Science. 1992 Jun 5;256(5062):1416-9
1604315
Protein Sci. 2005 Jun;14(6):1438-46
15883184
Biochim Biophys Acta. 2010 Feb;1800(2):96-106
19647786
J Biol Chem. 2004 Dec 24;279(52):54103-9
15494402
Diabetes. 1999 Dec;48(12):2407-13
10580430
J Biol Chem. 1992 May 5;267(13):9005-13
1533623
Methods Enzymol. 2006;415:113-33
17116471
Mol Biol Cell. 2008 Oct;19(10):4130-40
18653473
J Protein Chem. 2003 Apr;22(3):249-58
12962325
Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13793-8
18779572
J Biol Chem. 2008 Dec 5;283(49):33935-41
18840611
EMBO J. 2001 Nov 1;20(21):5863-75
11689427
Cell. 2000 Sep 1;102(5):615-23
11007480
Mol Biosyst. 2007 Nov;3(11):766-72
17940659
Genes Dev. 2005 Sep 15;19(18):2199-211
16166381
Trends Biochem Sci. 2006 Jun;31(6):342-8
16679021
Nature. 2007 Apr 26;446(7139):1017-22
17460662
Science. 2000 Aug 11;289(5481):905-20
10937989
Nat Chem Biol. 2010 May;6(5):338-43
20305658
Science. 2000 Aug 11;289(5481):920-30
10937990
J Mol Biol. 1973 Feb 19;73(3):329-49
4686199
FEMS Yeast Res. 2004 Dec;5(3):271-80
15556089
Anal Chem. 2008 Dec 15;80(24):9526-33
19007248
J Biol Chem. 2007 May 11;282(19):14056-64
17360704
J Biol Chem. 2008 May 9;283(19):13009-20
18353774
J Biol Chem. 2002 Jan 18;277(3):1755-61
11788610
Science. 2000 Jun 16;288(5473):2045-7
10856218
Curr Biol. 2007 May 1;17(9):749-60
17446074
Methods Enzymol. 1979;59:402-10
440082
Cell. 2005 Oct 7;123(1):49-63
16213212
Biochem Soc Trans. 2008 Aug;36(Pt 4):653-7
18631135
Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7029-36
11416183
Mol Cell Proteomics. 2002 Oct;1(10):791-804
12438562
Methods Enzymol. 1979;59:502-15
440085
Anal Bioanal Chem. 2008 Apr;390(8):2089-97
18369606
Annu Rev Biochem. 2008;77:177-203
18518820
Transfus Clin Biol. 1999 Feb;6(1):7-16
10188208
Proc Natl Acad Sci U S A. 1986 Oct;83(19):7137-41
2429301