Functional and structural investigation of Drosophila UNC- 45, a chaperone for myosin

The UCS family of proteins is important for myosin folding, stability, and maintenance. To understand the expression pattern, function, and structure of a UCS protein, the Drosophila UNC-45 (dUNC-45) was investigated. In chapter one, Western blot analysis indicates that dUNC-45 is expressed throughout development. Immunofluorescence confocal microscopy shows strong dUNC-45 expression in the body-wall muscles of 14 hour wild-type embryos. Examination of third instar wild-type larvae body-wall muscles using the same approach localized dUNC-45 to the Z discs of sarcomeres. In the dUNC-45 knock out line (T-33), the strong dUNC-45 skeletal muscle expression is lost in the 14 hour old embryos and the embryos do not hatch. Electron microscopy assessment of 22 hour T-33 embryos showed poor myofibril organization and a loss of thick filaments, which is reflected by a loss of myosin on the Western blot analysis. Results in the wild-type and the T- 33 embryos suggest a post-translational association between myosin and dUNC-45. In chapter two, the chaperone function of bacterially expressed dUNC-45 was analyzed using in vitro chaperone assays. The results demonstrate that dUNC-45 is capable of refolding chemically denatured citrate synthase (CS) and suppressing heat-induced aggregation of CS, [alpha]-lactalbumin, and myosin. The addition of ATP or AMP-PNP enhanced dUNC-45 chaperone function, but no ATP hydrolysis was detected. in vivo, immunofluorescence confocal microscopy of third instar wild-type larvae body-wall muscle suggest that heat stress induces translocation of dUNC-45 from the Z disc to the A band and possibly up-regulates protein level as shown in Western analysis. Chapter three focuses on the derivation and analysis of dUNC-45 x-ray crystal structure using bacterially expressed dUNC-45. Our 3.0 Å resolution model shows the Central and the UCS domains of dUNC-45 are composed of armadillo repeat protein motifs, but that the TPR domain is not resolved. Inspection of the surface hydrophobicity unveiled a groove in the UCS domain as the possible active site for myosin interaction. Future studies will involve defining the structure-function relationship between particular domains of UNC-45 and its chaperone activit