Tubulin, the subunit protein of microtubules, consists of an alpha and beta subunit, each of which exists as various isotypes, differing from each other in their amino acid sequences and their post-translational modifications. The fundamental aim of this proposal is to test the hypothesis that these differences are functionally significant. We shall focus on the various beta isotypes and on two of the post-translational modifications undergone by the tubulin molecule. The function of one of these, the phosphorylation of betaIII-tubulin, is not known. The other modification, the covalent addition of glutamate residues to the gamma- carboxyl groups of glutamates in alpha and betaIII, is not understood, and is unique among known proteins. We have used monoclonal antibodies to purify tubulin isotypes and found that they exhibit dramatic differences among themselves in their assembly and drug-binding properties, and their cellular distributions. We have also made the first accurate measurements of phosphate content of individual tubulin isotypes and found that only betaIII, and the alpha and binding to betaIII, are phosphorylated. In this proposal, we intend to carry out the following specific aims: 1. To determine the role of tubulin phosphorylation by locating the phosphate groups in the primary structure of tubulin, removing the phosphates, preparing antibodies capable of distinguishing between phosphorylated and non-phosphorylated tubulin, and separating these two forms of tubulin to determine their properties in vitro. 2. To examine the interaction of tubulin isotypes with proteins involved in microtubule assembly and function. For each form of tubulin, we shall measure its binding to, and its effects on the activities of, proteins mediating microtubule function, such as kinesin and cytoplasmic dynein. We shall distinguish the effects of the various tubulin isotypes and of phosphorylated and non- phosphorylated tubulin. 3. To determine the cellular and subcellular distributions of the beta isotypes and of phosphorylated and non- phosphorylated tubulin. We shall use monoclonal antibodies to determine the distributions of the various forms of tubulin in cells. We shall see how the apparent function of the microtubule in vivo, whose isotype composition and state of phosphorylation we will determine, is correlated with the known functions of those isotypes in vitro. 4. To identify and purify the enzymes involved in the glutamylation of tubulin. The purification of the enzymes that carry out this unique modification will be a major step toward understanding the role of glutamylation.
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