Structural characterization by tandem mass spectrometry of the posttranslational polyglycylation of tubulin

Polyglycylation is a posttranslational modification specific to tubulin. Th is modification was originally identified in highly stable microtubules fro m Paramecium cilia. As many as 34 posttranslationally added glycine residue s have been located in the C-terminal domains of Paramecium alpha- and beta -tubulin. In this study, post source decay matrix-assisted laser desorption /ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ES I Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta -tubulin, from either dynamic cytoplasmic microtubules or stable axonemal m icrotubules, can be glycylated on each of the last four C-terminal glutamat e residues Glu(437), Glu(438), Glu(439), and Glu(441) in the sequence (427) DATA- EEEGEFEEEGEQ(442). In both dynamic and stable microtubules the most a bundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu(437) and Glu(438) and one glyci ne residue on both Glu(439) and Glu(441). Th, number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal mi crotubules were compared by MALDI MS.(1) The abundance of the major glycyla ted isoforms in axonemal tubulin decreases regularly with glycylation level s from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycyl ation levels from 6 to 9. However, the polyglycine chains are similarly dis tributed on the four C-terminal glutamate residues of cytoplasmic and axone mal tubulin. The polyglycylation results in bulky C-terminal domains with n egatively charged surfaces, ail surrounding the microtubular structure.