Most higher eukaryotic tubulins are separated into a-and J3-tubulin when electrophoresed in NaDodSO4-denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80). By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two a-tubulins and one P-tubulin. Moreover, at least five A-and two .3-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Three of these a isotypes and one (3 isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro. Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related. Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.

Ciliary and cytoplasmic tubulins were isolated from Tetrahymena thermophila [strain SB 71 1'1 and analyzed by 2-D sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by I-D peptide mapping in order to begin to determine whether the assembly of a specific microtubule-containing organelle is regulated by the availability of a specific tubulin. Separation of Tetrahyrnena tubulins by 1-D SDS-PAGE' was a function of the pH of the separating gel with maximum resolution of a and j3 tubulins occurring at pH 8.25-8.50. The a subunit was identified as the fastest migrating tubulin under these conditions. In I-D separating gels with a pH greater than 8.7, the a and j3 tubulins merged into a single band that comigrated with chick and bovine brain j3 tubulin.High resolution isoelectric focusing (IEF) and 2-D SDS-PAGE3 indicated that the ciliary axoneme was composed of a major a (a3) and two minor a tubulins (al, a2), and a major j3 (p2) and a minor j3 (PI) tubulin. The minor axonemalp, was found to be the major ( 3 tubulin in solubilized central pair microtubules, obtained by dialysis against Tris-EDTA! By contrast, p2 was the major j3 tubulin in outer doublet microtubules.Tetrahymena cytoplasmic tubulin purified and assembled in vitro' and was composed of two unique tubulin subunits, a4 and a5 that did not comigrate with any of the axonemal tubulins and a j3 tubulin that comigrated with axonemal p2. The major cytoplasmic a, a', was the fastest migrating on SDS-PAGE and had the most basic PI on IEF gels of all the tubulin subunits.Axonemal and cytoplasmic a and j3 tubulins, resolved by I-D SDS-PAGE, were cut out of these gels, and compared with one another after 1-D limited proteolytic digestion with Staphylococcus aureus V8 protea~e.~?' The resulting peptides were analyzed by electrophoresis in pH 8.25 and 8.80 separating gels. No differences were observed in the peptide maps of axonemal and cytoplasmic j3 tubulin at either pH 8.25 or 8.80. By contrast, the pattern of peptides resolved from cytoplasmic a tubulin at pH 8.25 was different than the axonemal a cleavage products. The peptide maps of axonemal and cytoplasmic a tubulins were identical in pH 8.80 gels.In summary, we have identified seven organelle-specific tubulins by IEF, I-D and 2-D SDS-PAGE, and by 1-D peptide mapping (see TABLES 1 and 2). It is likely that TABLE 1. Isoelectric Points of Tubulin Subunits Subunit P 1 . 2 ff1,2.3 ff4 ff3 PI 5.1-5.2 5.3-5.4 5.5 5.55 132