Expression of a human alpha-tubulin: properties of the isolated subunit.
We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer.
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000302:isolation & purification; D017239:Paclitaxel; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014357:Trypsin; D014404:Tubulin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D000470:Alkaloids; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D010446:Peptide Fragments; D014404:Tubulin; D002918:Chymotrypsin; D014357:Trypsin; D017239:Paclitaxel