Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.
We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)
Eddé
B
B
Laboratoire de Biochimie Cellulaire, Collège de France, Paris.
Rossier
J
J
Le Caer
J P
JP
Promé
J C
JC
Desbruyères
E
E
Gros
F
F
Denoulet
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain; D002478:Cells, Cultured; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D011099:Polyglutamic Acid / Q000737:chemistry / Q000378:metabolism*; D014404:Tubulin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D014404:Tubulin; C039520:tyrosine-tubulin; D011099:Polyglutamic Acid; D014443:Tyrosine