Translation of beta-tubulin mRNA in vitro generates multiple molecular forms.
We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.
Yaffe
M B
MB
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.
Farr
G W
GW
Sternlicht
H
H
D000818:Animals; D001921:Brain / Q000378:metabolism; D002645:Chickens; D002918:Chymotrypsin; D004247:DNA / Q000235:genetics; D006160:Guanosine Triphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014404:Tubulin / Q000235:genetics* / Q000302:isolation & purification
D046911:Macromolecular Substances; D008868:Microtubule Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D014404:Tubulin; D006160:Guanosine Triphosphate; D004247:DNA; D002918:Chymotrypsin