Abstracts Biological Sciences

Effect of mutations in DUNC-45 on its activity as a myosin chaperone, using Drosophila as a modelm

by Lakshmi Milind Prabhu

Myosin II is an actin-based molecular motor that drives movement and its folding is a critical step in myofibril formation and muscle assembly. Myosin folding is mediated by the muscle specific chaperone UNC-45. UNC-45 has a C-terminal UCS domain that binds to the myosin head. UNC-45 is important for myosin folding and protection against stress conditions. Interaction between the myosin head domain and the UCS domain of UNC-45 is well established. However, the precise residues on each of these proteins that interact with each other are not well known. The UCS domain is a highly conserved region across all isoforms of UNC-45. In this study, three residues in the UCS domain: arginine 864, asparagine 786 and serine 747, were chosen to test for their importance in myosin interaction and were mutated to alanine, arginine and arginine, respectively. These three residues lie in the conserved cleft of Drosophila melanogaster UNC-45 (DUNC-45) and they are oriented outwards on the protein surface. We hypothesized that these residues mediate interactions of the DUNC-45 chaperone with the myosin head. Molecular cloning of the constructs in bacteria permitted induction of mutant protein expression and these were purified using affinity and size exclusion chromatography. They were tested for chaperone function and stability using biochemical assays. Aggregation kinetics showed the mutant proteins were quite heat-sensitive, suggesting that the amino acid residues may be important for DUNC-45 function. Myosin-binding assays showed no difference in the ability of the mutant UNC-45 molecules to bind myosin compared to WT-DUNC-45. The R864A-DUNC-45 mutation was also expressed in D. melanogaster using P element-mediated germline transformation. Ten viable fly lines were obtained. Protein was expressed in the DUNC-45 null background, to ensure that only the mutant DUNC-45 is expressed in the transgenic flies. Functional tests were performed to analyze the effect of this mutation on the function of the myosin protein in vivo. Flight testing of one-day-old flies revealed no significant difference between the transgenic mutant flies and the control. No change in myosin heavy chain accumulation during adult thoracic muscle development was observed in the presence of the mutated chaperone by SDS-PAGE gel analysis. Thus, flies grown at room temperature were unaffected by the mutant DUNC-45. The effect of increased temperature on development was also tested. Even though the lethality of heat-shocked embryos was slightly higher for the mutant transgenic lines compared to control, the difference was not statistically significant. Overall, in vitro studies suggested that the R864A DUNC-45 was prone to aggregation but did not significantly affect myosin accumulation or function in vivo. In summary, in vitro aggregation studies revealed that these mutant proteins could be important for regulating chaperone function, however binding studies of all three mutants and in vivo studies of the R864A-DUNC45 mutant did not reveal a significant effect of the…