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 A 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 activity.