STRUCTURAL AND GENETIC ASPECTS OF TUBULIN INTERACTIONS

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Microtubules are multisubunit protein structures found in all eukaryotic cells which are involved in a variety of cell functions. They are composed primarily of two genetically related proteins of molecular weight 50,000 termed alpha and beta tubulin. Tubulins isolated from lower eukaryotes such as yeast, fungi, and protozoa (which may differ by as much as 30% in amino acid sequence from mammalian tubulins) exhibit greatly different assembly, stability, and ligand binding properties than tubulins isolated from mammalian brain. The long term goal of our research is to use genetic and biophysical techniques to determine how the structural and functional properties of microtubules relate to the amino acid sequence of the alpha and beta tubulins and to define functional domains of the tubulin monomers and protomers. The specific aims of this proposal are: 1. To prepare cloned tubulins of a protozoan (African trypanosome) and higher eukaryote brain (chicken), develop conditions for reconstitution of biologically active tubulin protomers, and compare the assembly, conformation, and ligand binding properties with authentic trypanosome and brain tubulin. 2. To purify and characterize authentic trypanosome tubulin in terms of its assembly, stability, and susceptibility to antimitotic agents such as colchicine and the benzimidazoles. 3. To define the molecular mechanisms of interactions of guanine nucleotides and antimitotic agents by assembly and characterization of interspecies dimers or genetically altered (chimeras and site-specific mutants) alpha and beta tubulins. 4. To determine whether the trypanosome or chick brain beta tubulins (or regions of them) are biologically functional in yeast. An understanding of the mechanisms by which guanine nucleotides interact with tubulin and modulate its properties could contribute significantly to our understanding of its role in other important biological systems such as the G proteins, ras oncogenes, and ribosome. Knowledge of the factors governing interaction of antimitotic agents with tubulin and their related effects on microtubule structure could lead to the development of new antimitotic agents useful for treatment of protozoan diseases such as trypanosomiasis or perhaps malignancies.