Both Carboxy-Terminal Tails of α- and β-Tubulin Are Essential, but Either One Will Suffice

contains unique, essential sequence elements, i.e., they Microtubules (MTs) are organized into distinct syshave completely redundant functions. tems essential for cell shape, movement, intracellular Glycylation is known to occur on glutamate residues transport, and division [1]. Electron crystallographic in the C terminus of and -tubulin in cells with axoanalyses [2] provide little information about how MTs nemes. Tetrahymena cells can tolerate elimination of produce diverse structures and functions, perhaps be-tubulin glycylation, but large reductions in -tubulin cause they failed to visualize the last 10 residues of glycylation are lethal [10]. Also, lethal mutations, with the and the last 18 of the -tubulin C-terminal tails multiple -tubulin glycylation sites changed from E to (CTTs) [3], which likely play a role in MT diversity. CTTs D, could be rescued by replacing the last 6 residues define conserved, nonallelic isotypes in mammals [4], of -tubulin with the last 8 residues of -tubulin [10]. are major sites of posttranslational modifications Analysis of glycylation levels on and -tubulin in these (PTMs) [5–7], are binding sites for microtubule-associmutants suggested this was a quantitative effect, reated proteins (MAPs) [3], and determine MT motor flecting the fact that the C terminus is more highly processivity [8]. Using mutagenesis and homologous glycylated than the C terminus [10]. gene replacement in Tetrahymena thermophila, we Glutamylation also occurs on glutamate residues in analyzed mutations, deletions, tail switches, and tail the CTTs of many tubulins [5–7], including Tetrahymena duplications of and -tubulin CTTs. We demon[11], where it occurs on both and -tubulins (our strate that a tail is required for the essential function unpublished data). To determine whether the essential of both and -tubulin. However, the two tails are function of the C terminus was related to either glutainterchangeable, and cells grow normally with either mylation or glycylation, we constructed the BTU1-9D an or a tail on both tubulins. In addition, an gene mutation with all possible glycylation and glutamylation containing a duplicated C terminus rescues a lethal sites in the BTU1 C terminus mutated from Es to Ds. As mutant lacking all known posttranslational modificaexpected, this mutation failed to produce viable progtion sites on the C terminus but cannot rescue deleeny when transformed into mating BTU1/BTU2 knocktion of the tail. Thus, tubulin tails have a second out heterokaryons. However, this lethal mutation could essential function that is not associated with postbe rescued by cotransformation with ATU1 A1 and translational modification. ATU1 A2, constructs in which the ATU1 gene con-