Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro.
A post-translational modification of tubulin with potential regulatory significance has been revealed by the discovery of an enzyme (tubulin-tyrosine ligase) in brain extracts which can add a tyrosine residue to the alpha chain, apparently through peptide bond linkage to a C-terminal glutamate. We have investigated whether this modification also occurs in vivo, and whether it alters the extent to which tubulin can assemble in vitro. Cytoplasmic tubulin purified from bovine brain by cycles of assembly was shown to be partially tyrosylated. Carboxypeptidase A digestion of isolated alpha chains liberated about 0.3 equivalent of tyrosine. Brief digestion of native tubulin increased the proportion of alpha chains which could be tyrosylated by ligase, from 25 to 45%. The tubulin assembled to the same extent before and after carboxypeptidase treatment. When tubulin was purified after introducing labeled tyrosine with ligase, the labeled species assembled in the same proportion as unlabeled. Thus tubulin can be incorporated into microbubules in vitro with or without C-terminal tyrosine. An apparent resolution of alpha chain into two components by hydroxylapatite chromatography was shown not to be due to the presence or absence of C-terminal tyrosine. Tubulin-tyrosine ligase was found in extracts of every rat tissue examined, but was not detected in sea urchin eggs before or after fertilization, in Tetrahymena cells or cilia, or in yeast. Cultured neuroblastoma cells fixed tyrosine into tubulin alpha chains under conditions where protein synthesis was inhibited; this in vivo fixation appeared to be into an insoluble moiety of tubulin. Incidental to these studies, a new assay utilizing an enamine substrate for carboxypeptidase was investigated.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010063:Ovum / Q000201:enzymology; D051381:Rats; D012440:Saccharomyces / Q000201:enzymology; D012617:Sea Urchins; D013768:Tetrahymena / Q000201:enzymology; D014404:Tubulin / Q000032:analysis / Q000378:metabolism*; D014443:Tyrosine / Q000032:analysis / Q000378:metabolism*
D006023:Glycoproteins; D014404:Tubulin; D014443:Tyrosine; D008025:Ligases