Polyglutamylation of nucleosome assembly proteins

Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units a nd leading to a very unusual protein structure. A monoclonal antibody direc ted against glutamylated tubulin (GT335) was found to react with other prot eins present in HeLa cells. After immunopurification on a GT335 affinity co lumn, two prominent proteins of similar to 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 t he deposition of core histone complexes onto chromatin, Strikingly, NAP-1 a nd NAP-2 were found to be substrates of an ATP-dependent glutamylation enzy me copurifying on the same column. We took advantage of this property to sp ecifically 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 g lutamyl units, respectively. Two putative glutamylation sites were localize d 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 tubul in are polyglutamylated and open new perspectives for studying NAP function .