Polyglutamylation of Nucleosome Assembly Proteins

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 ϳ50 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 copurifying 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.

The structure of nucleosome assembly protein 1

“…Roughly 50% of all NAP-1 molecules are glutamylated in HeLa cells. In contrast, only Ϸ4% of tubulin (the only other protein known to be glutamylated) is found in glutamylated form in the same cell (39). Two glutamylation sites (modified with up to 10 glutamyl units) were identified in the C-terminal region of NAP-1 and NAP-2 in HeLa cells.…”

“…Two glutamylation sites (modified with up to 10 glutamyl units) were identified in the C-terminal region of NAP-1 and NAP-2 in HeLa cells. This modification may lead to the formation of a second or third acidic C-terminal moiety (39). Accumulation of glutamyl moieties would allow for modulation of the total negative charge of the C-terminal acidic domain, in a reversible manner, with the potential of switching between different NAP functions.…”

TTLL10 is a protein polyglycylase that can modify nucleosome assembly protein 1

“…359 and 360 in the C-terminus of NAP1 NAP1 can be glutamylated at Glu-356 and -357 in the C-terminal Glu-rich regions [3]. To investigate which Glu residues are required for polyglycine ligation to NAP1, we generated various mutants of NAP1 (Fig.…”

“…Proteins other than tubulin can also undergo these modifications. Nucleosome assembly proteins (NAPs) are subjected to glutamylation [3], and 14-3-3 proteins [4] and Hsp70/ Grp170-related protein [5] can be glycylated.…”

The Tubulin Code

“…42,43 Both of these modifications are relatively rare although non-tubulin targets have recently been identified. [44][45][46] In mammals, tubulin glycylation is mostly restricted to axonemes of motile cilia and flagella [47][48][49] whereas glutamylation is abundant in neurons, on centrioles, in axonemes, and in spindle microtubules. 27,50,51 In ciliated protists, both polymodifications are found in numerous microtubular networks, including cytoplasmic and axonemal microtubules.…”