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In Tetrahymena Thermophila Jacek Gaertig,* Manuel A

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In Tetrahymena Thermophila Jacek Gaertig,* Manuel A Acetylation of Lysine 40 in ot-tubulin Is Not Essential in Tetrahymena thermophila Jacek Gaertig,* Manuel A. Cruz, Josephine Bowen, Long Gu, David G. Pennock,* and Martin A. Gorovsky Department of Biology, University of Rochester, Rochester, New York 14627; and *Department of Zoology, Miami University, Oxford, Ohio 45056 Abstract. In Tetrahymena, at least 17 distinct microtu- acetylated tubulin was detectable in these transform- bule structures are assembled from a single primary se- ants using a monoclonal antibody specific for acety- quence type of oL- and 13-tubulin heterodimer, preclud- lated lysine 40. Surprisingly, mutants lacking detectable ing distinctions among microtubular systems based on acetylated tubulin are indistinguishable from wild-type tubulin primary sequence isotypes. Tetrahymena tubu- cells. Thus, acetylation of o~-tubulin at lysine 40 is non- lins also are modified by several types of posttransla- essential in Tetrahymena. In addition, isoelectric focus- tional reactions including acetylation of a-tubulin at ing gel analysis of axonemal tubulin from cells unable lysine 40, a modification found in most eukaryotes. In to acetylate o~-tubulin leads us to conclude that: (a) Tetrahyrnena, axonemal o~-tubulin and numerous other most or all ciliary ot-tubulin is acetylated, (b) other microtubules are acetylated. We completely replaced lysines cannot be acetylated to compensate for loss of the single type of a-tubulin gene in the macronucleus acetylation at lysine 40, and (c) acetylated o~-tubulin with a version encoding arginine instead of lysine 40 molecules in wild-type cells contain one or more addi- and therefore cannot be acetylated at this position. No tional charge-altering modifications. ICROTUBULES are involved in cytoplasmic organi- types cannot be substituted by transgenic expression of a zation and motility in eukaryotic cells. They different tubulin isotype (21, 33). M form a large variety of distinct organelles includ- Because most cell types contain a mixture of different ing a cytoplasmic network, the mitotic spindle, the cen- tubulin isotypes and multiple, distinct microtubule sys- trosome, cilia, and flagella. The core component of micro- tems, tubulin isotype specialization could extend to the tubules is a heterodimer of et- and J3-tubulin proteins. In subcellular level. However, in most studies, cytoplasmic most organisms tubulins occur in multiple isoformsJ In microtubules were found to contain a mixture of all tubu- lower eukaryotes such as Saccharomyces cerevisiae, As- lin isotypes expressed and thus no selective partitioning of pergillus nidulans, Dictyostelium discoideum and Tetrahy- endogenous tubulin isotypes was found (21, 29, 30, 33). Al- mena, a relatively low number of tubulin isogenes is though a few cases of preferential partitioning of tubulin mainly involved in coordinate regulation of tubulin abun- isotypes have been found either within the cell or in an in dance during vegetative growth and development (1, 7, 15, vitro polymerization assay (4, 9, 38), there is no direct evi- 25, 34, 45, 50). In multicellular organisms, several tubulin dence that these phenomena reflect intracellular partition- isogenes are usually expressed and at least some tubulin ing of tubulin isotypes with specialized intracellular func- isotypes 1 appear to perform specialized functions. For ex- tions. ample, in Drosophila, functions of at least two tubulin iso- In most cell types, the primary tubulin gene products are further modified by secondary modifications (18, 36). Posttranslational tubulin modifications (PTM) 2 usually af- Address all correspondence to Martin A. Gorovsky, Dept. of Biology, fect only a subset of the microtubules in a cell. It is intrigu- University of Rochester, Rochester, NY 14627. Tel. (716) 275-6988. Fax: ing that a small number of unicellular organisms which do (716) 275-2070. not have detectable PTMs, such as fungi or Dictyostelium 1. Dr. Gaertig's present address is Dept. of Cellular Biology, The Univer- discoideurn, also have relatively simple microtubular sys- sity of Georgia, Athens, GA 30602-2607. Throughout this paper the term tems consisting largely of spindles and cytoplasmic micro- isoforms will be used to refer to different protein products of a single type tubules (2, 50). This observation raises the possibility that, (a and 13) of tubulin that differ in electrophoretic mobility. The term iso- types will refer to the protein products of different (ct and 13) tubulin genes. Isoforms can be different secondarily modified products of a single 2. Abbreviation used in this paper: PTM, posttranslational tubulin modifi- (a and 13) tubulin gene or can be derived from different genes. cation. © The Rockefeller University Press, 0021-9525/95/06/1301/10 $2.00 The Journal of Cell Biology, Volume 129, Number 5, June 1995 1301-1310 1301 in organisms which express complex microtubular or- izing drug taxol. The mutation responsible for this phenotype is an alanine ganeUes, PTMs act in concert with mechanisms controlling to threohine replacement at position 65 in the ~-tubulin gene that will be described in detail elsewhere (J. Gaertig, D. Pennock, and M. Gorovsky, tissue-specific isotype transcription to execute diverse unpublished results). Tetrahymena were grown in SPP medium (17) with functions of microtubules. shaking at 30°C. Antibiotic-containing SPPA medium (12) was used for In general, a positive correlation exists between the sta- transformation, plating, and growth tests. bility of microtubule subpopulations and the extent of For growth experiments, 50 ml SPPA cultures in 250 ml Erlenmeyer their secondary modification (16, 24, 28, 42, 51). However, flasks were inoculated to contain 104 cells/ml. Cells were grown at 30°C without shaking and periodically counted (Coulter Counter, model ZF; despite intense study, the functional importance of even a Coulter Electronics, Hialeah, FL). In some growth experiments cells were single type of PTM remains unknown. grown at different temperatures or with addition of NaCI or sucrose to the One of the best characterized PTMs is the acetylation of culture medium to test for phenotypic effects of tubulin acetylation. In the e~-tubulin at a conserved lysine at position 40 (10, 27). Al- drug response experiments, cells were incubated in variable drug concen- trations in 200 ~1 of SPPA per well of a 96-well microtiter plate at 30°C, though this modification is frequently associated with sta- starting at 104 cell/ml density and counted after 24 h. ble microtubules in vivo, it was found that isolated acety- To measure the swimming rate, cells from growing cultures were ob- lated and unacetylated microtubules have similar stabilities served using a 40× lens under an Olympus BH2 microscope and photo- in vitro (32). Thus, acetylation does not directly stabilize graphed by exposing Kodak T-Max 400 film for 1 s. The negatives were microtubules. To test the function of ot-tubulin acetylation projected using a photographic enlarger and paths of swimming cells were measured with a map reader. The absolute distances were calculated by in vivo, Kozminski et al. (26) expressed a mutant non- calibrating with a stage micrometer. acetylatable a-tubulin gene in Chlamydornonas. Up to To test pairing efficiency (39), the transformed clones were starved for 70% of endogenous tubulin was replaced without causing 24 h in 10 mM Tris-HCl, pH. 7,5. Cells (10 ml at a density 2 x 105 cells per any detectable phenotypic alteration. However the 30% of ml) of each tested strain were mixed with an equal number of untrans- formed wild-type (CU428) cells and incubated at 30°C. Samples of cells wild-type acetylatable a-tubulin still present in these were withdrawn periodically, fixed with 1% (vol/vol) trichloroacetic acid transformed Chlamydomonas cells could have been suffi- and the fraction of total cells in pairs was determined. cient to support the normal functions of acetylated micro- tubules. Immunodetection of Acetylated lys 40 Tubulin Epitope In there is only a single type Tetrahymena thermophila Monoclonal antibody 6-11B-l, which is specific for a conserved epitope of et-tubulin gene (7, 35) and two genes that encode identi- surrounding the acetylated lysine at residue 40 on a-tubulin in Chlamydo- cal [3-tubulin proteins (15). Thus, all of the (at least) 17 dif- monas (41), was used to detect acetylated a-tubulin. This antibody was ferent microtubule systems in this ciliated protozoan are generously provided by Dr. O. Piperno (Rockefeller University, New assembled using a single type of tubulin dimer primary se- York, NY). As a general anti-a-tubulin probe we used the DM1A mono- clonal antibody (Amersham Corp., Arlington Heights, IL). Immunofluo- quence, providing one of the most striking examples of the rescent detection of tubulins was performed as described previously (11) multifunctionality of a single tubulin primary sequence. except that 1 I~M taxol was added to the cytoskeleton extraction buffer to However, Tetrahymena tubulins are secondarily modified help retain the cytoskeleton in the extracted cells. The 6-11B-1 and by several mechanisms, including acetylation, polyglutamy- DM1A antibodies were used at 1/5 and 1/500 dilution, respectively. lation (6, 40, 47) and possibly phosphorylation (20), to pro- duce numerous tubulin isoforms (47). Thus, Tetrahymena Plasmid Vectors provides an excellent system to study the function of The pTUB100 plasmid contains the 3.2-kb HindlII genomic fragment of PTMs in the absence of any complicating genetically en- the ct-tubulin gene of T. thermophila (35). pTUB100E3 (see Fig. 2) is a de- coded tubulin variation. rivative of pTUB100 in which a HaelII restriction site has been intro- duced by a silent substitution at alanine codon 281 (GCT to GCC). The Recently, we described a method for homologous re- pTUB-R40 is a derivative of pTUB100E3 where an arginine codon at po- placement of one of the two [3-tubulin genes in Tetrahy- sition 40 was introduced in place of the wild-type lysine codon (AAG mena (14) in which a wildtype taxol resistant gene replaces codon to AGA).
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