Structure of the polyglutamyl side chain posttranslationally added to alpha-tubulin.
Polyglutamylation, a new posttranslational modification of tubulin identified originally on the acidic alpha variants by Eddé et al. (Eddé, B., Rossier, J., Le Caer, J. P., Desbruyeres, E., Gros, F., and Denoulet, P. (1990) Science 247, 83-85), consists of the successive addition of glutamyl units to the Glu445. To characterize their linkage mode mouse tubulin was posttranslationally labeled with [3H]glutamate. After digestion of [3H]tubulin with thermolysin, up to eight radioactive peaks were separated on an anion exchange column (DEAE). Combined use of Edman degradation sequencing and mass spectrometry analysis of the first 6 one indicated that they all correspond to the same COOH-terminal sequence 440VEGEGEEEGEE450 bearing one to six glutamyl units on the Glu445. The first glutamyl residue is amide-linked to the gamma-carboxyl group of Glu445, but the additional residues can be linked to the gamma- or alpha-carboxyl groups of the preceding one. All possible linkages for the biglutamylated tubulin peptides (gamma 1 alpha 2, gamma 1 gamma 2) and triglutamylated (gamma 1 alpha 2 alpha 3, gamma 1 alpha 2 gamma 3, gamma 1 alpha 2 gamma 2, gamma 1 gamma 2 alpha 3, gamma 1 gamma 2 gamma 3) were synthesized. These different peptides were successfully separated on a C18 5-micron reverse phase column. We found that the bi- and triglutamylated tubulin peptides behave as the gamma 1 alpha 2 and gamma 1 alpha 2 alpha 3 synthetic peptides, respectively. These results indicate that the second and third glutamyl residues of the polyglutamyl side chain are amide-linked to the alpha-carboxyl group of the preceding unit.
Redeker
V
V
Laboratoire de Physiologie Nerveuse, Centre National de Recherche Scientifique, Gif-sur-Yvette, France.
Le Caer
J P
JP
Rossier
J
J
Promé
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D011099:Polyglutamic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D011099:Polyglutamic Acid