Abstract
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.
Highlights
Tubulin, the main microtubule component, is subjected to several posttranslational modifications
Western blotting analysis of HeLa cell soluble extract with GT335, a mAb directed against the polyglutamylated motif of tubulin [28], revealed several reactive proteins of 48 –52 kDa that fall within the region of migration of ␣- and -tubulin
Since the discovery of polyglutamylation, there has been a question as to whether this posttranslational modification is restricted to tubulin
Summary
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. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu357 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. In vitro blot overlay assays strongly suggest that the polyglutamyl side chain of tubulin can regulate differentially the binding of various microtubule-associated proteins, in a manner dependent on the length of the side chain [16, 17]. Examination of other cell types and organisms indicates that polyglutamylation of NAPs appears as a general phenomenon These results provide new insights for studying the regulation of NAP function. They suggest that polyglutamylation might be a more general posttranslational modification than previously believed
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