Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules inParamecium

Abstract

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono-and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the ␣-and ␤-tubulin subunits are modified by only 1-5 and 2-9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases

“…The reversible nature of polyglycylation and the existence of tubulin deglycylases were previously shown in cytoplasmic extracts of the ciliate Paramecium tetraurelia, but the enzymes were not identified (6). gDIP1 and gDIP2 belong to the MH clan, subfamily M20C.…”

“…We demonstrated that the g14-3-3 is modified in a peculiar fashion by the phosphorylation of Thr-214 and the polyglycylation of Glu-246 (3,4). The glycylation, first discovered at the C-terminal domain of ␣-and ␤-tubulin, is a post-translational modification consisting of the covalent addition of one or multiple glycines to the ␥-carboxyl groups of specific glutamic acids of target proteins (6,7). Recently, polyglycylation has been also reported for several proteins, including the mammalian nucleosome assembly proteins (8 -10).…”

Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila

“…The GT335 MAb recognizes a branch point of a glutamyl side chain consisting of at least one glutamate on either ␣-or ␤-tubulin (63) (see the footnote to Table S1 in the VOL. 7,2008 TTLL1 AND TTLL9 GLUTAMYLATE ␣-TUBULIN 1363 supplemental material). GT335 labeled all of the microtubule locations in vegetative cells ( Fig.…”

“…7,2008 TTLL1 AND TTLL9 GLUTAMYLATE ␣-TUBULINTetrahymena strain, ATU1-6D, in which all six glutamates within the CTT of Atu1p (the only conventional ␣-tubulin in Tetrahymena) (38) were replaced with aspartates to preserve the charge but prevent polymodifications. The ATU1-6D cells are viable and motile, indicating that polymodifications on ␣-tubulin are not essential.…”

“…With the reduced efficiency of basal body maturation, the rows of basal bodies could shorten and some rows could even be lost VOL. 7,2008 TTLL1 AND TTLL9 GLUTAMYLATE ␣-TUBULIN 1369 (10,11,18). Within a growing wild-type population, a vast majority of cells maintains the number of ciliary rows within the so called "stability range" of 18 to 20 (17,40).…”