Acetylation of Lysine 40 in ot-tubulin Is Not Essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of oLand 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. In Tetrahyrnena, 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. M ICROTUBULES 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 etand 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 isoAddress all correspondence to Martin A. Gorovsky, Dept. of Biology, University of Rochester, Rochester, NY 14627. Tel. (716) 275-6988. Fax: (716) 275-2070. 1. Dr. Gaertig's present address is Dept. of Cellular Biology, The University of Georgia, Athens, GA 30602-2607. Throughout this paper the term isoforms will be used to refer to different protein products of a single type (a and 13) of tubulin that differ in electrophoretic mobility. The term isotypes will refer to the protein products of different (ct and 13) tubulin genes. Isoforms can be different secondarily modified products of a single (a and 13) tubulin gene or can be derived from different genes. types cannot be substituted by transgenic expression of a different tubulin isotype (21, 33). Because most cell types contain a mixture of different tubulin isotypes and multiple, distinct microtubule systems, tubulin isotype specialization could extend to the subcellular level. However, in most studies, cytoplasmic microtubules were found to contain a mixture of all tubulin isotypes expressed and thus no selective partitioning of endogenous tubulin isotypes was found (21, 29, 30, 33). Although a few cases of preferential partitioning of tubulin isotypes have been found either within the cell or in an in vitro polymerization assay (4, 9, 38), there is no direct evidence that these phenomena reflect intracellular partitioning of tubulin isotypes with specialized intracellular functions. In most cell types, the primary tubulin gene products are further modified by secondary modifications (18, 36). Posttranslational tubulin modifications (PTM) 2 usually affect only a subset of the microtubules in a cell. It is intriguing that a small number of unicellular organisms which do not have detectable PTMs, such as fungi or Dictyostelium discoideurn, also have relatively simple microtubular systems consisting largely of spindles and cytoplasmic microtubules (2, 50). This observation raises the possibility that, 2. Abbreviation used in this paper: PTM, posttranslational tubulin modification. © The Rockefeller University Press, 0021-9525/95/06/1301/10 $2.00 The Journal of Cell Biology, Volume 129, Number 5, June 1995 1301-131