MUTAGENIC ANALYSIS OF YEAST ACTIN IN VIVO AND IN VITRO

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Our overall goal is to gain knowledge at the molecular level of factors contributing to actin stability, polymerizability, and the ability of actin to interact with the proteins that control its function within the cell. Besides being one of the two major components of the contraction producing machinery in muscle, actin is involved in a number of motile and contractile processes needed for cell function in non-muscle cells including cytokinesis, cell movement along a substratum, and cell shape determination. Defects in myosin, tropomyosin, and troponin, all proteins that interact with actin in the muscle sarcomere, have recently been shown to be responsible for some forms of cardiomyopathy. Understanding the mechanism underlying sarcomere malfunction in these disorders as well as understanding how the cell carries out and controls the motile processes with which it is involved requires an understanding of actin works at the molecular level. The recently solved X-ray crystal structure of actin has generated two untested hypotheses concerning actin function: 1) a 10 residue loop between actin subdomains 3 and 4 in an actin monomer in one helical strand inserts into a hydrophobic pocket in the opposite strand to produce a stabilization of the F-actin helix; 2) based on the similarity in tertiary structures of actin and HSC7O, H161, and Q137 are involved in the actin catalyzed hydrolysis of ATP, a reaction important for actin stability and filament dynamics. We will test these hypotheses using site- directed mutagenesis of yeast actin coupled with a yeast expression system to test in vivo and in vitro the effects of mutations in the hydrophobic loop and in H161 and Q137 on actin structure and function. Viable haploid cells containing only the mutant actin will be tested for growth at different temperatures, budding polarity will be assessed, and actin and chitin deposition will be analyzed by fluorescence microscopy. Mutant actins will be isolated in active form using DNAse I affinity chromatography and compared with wild-type yeast actin in terms of their thermostability, polymerizability, binding to and hydrolysis of ATP, and interaction with myosin. Using a mutant actin with an engineered reactive Cys near the hydrophobic loop we will label the -SH with fluorescent and spin label probes to monitor conformation changes in the loop region of both G and F-actin using fluorescence and EPR spectroscopy. We will test the effects of the loop mutations on filament stability using differential scanning microcalorimetry. Using reactive thiol affinity chromatography, we will establish a procedure for separating mutant from wild-type actin when expressed in the same cell. This will allow us to study mutant actins which by themselves are not compatible with yeast viability. These experiments will provide new information concerning the stabilization of F-actin and the role that ATP plays in actin filament dynamics and allow correlation of abnormal actin function in vitro with the phenotypes produced by these mutations in vivo.