Polyglutamylation of nucleosome assembly proteins.
Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.
Regnard
C
C
Biochimie Cellulaire, Collège de France, 11 place Marcelin Berthelot, 75005 Paris, France.
Desbruyères
E
E
Huet
J C
JC
Beauvallet
C
C
Pernollet
J C
JC
Eddé
B
B
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D018797:Cell Cycle Proteins; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011099:Polyglutamic Acid / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000276:immunology
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; C533557:NAP1L1 protein, human; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes; D010446:Peptide Fragments; D011506:Proteins; D014404:Tubulin; D011099:Polyglutamic Acid