Abstract. In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of oL-and 13-tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of a-tubulin at lysine 40, a modification found in most eukaryotes. InTetrahyrnena, axonemal o~-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of a-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of o~-tubulin at lysine 40 is nonessential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate o~-tubulin leads us to conclude that: (a) most or all ciliary ot-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated o~-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.
MICROTUBULES are involved in cytoplasmic organization and motility in eukaryotic cells. They form a large variety of distinct organelles including a cytoplasmic network, the mitotic spindle, the centrosome, cilia, and flagella. The core component of microtubules is a heterodimer of et-and J3-tubulin proteins. In most organisms tubulins occur in multiple isoformsJ In lower eukaryotes such as Saccharomyces cerevisiae, Aspergillus nidulans, Dictyostelium discoideum and Tetrahymena, a relatively low number of tubulin isogenes is mainly involved in coordinate regulation of tubulin abundance during vegetative growth and development (1,7,15,25,34,45,50). In multicellular organisms, several tubulin isogenes are usually expressed and at least some tubulin isotypes 1 appear to perform specialized functions. For example, in Drosophila, functions of at least two tubulin iso-