Muscle thick filament structure and assembly require precise determination of length and diameter, structural differentiation and nonequivalence of myosins, and dynamic regulation of assembly and disassembly in response to a variety of signals. Directed assembly of myosin, therefore, is more likely than simple self assembly of myosin into thick filaments (Epstein & Fischman, Science 251:1039, 1991). The
unc-45 locus has been proposed to encode a catalyst for thick filament assembly, based on the phenotype of the temperature sensitive (ts) strain CB286 (Epstein & Thomson, Nature 250:579, 1974). This locus has been shown to interact genetically with myosin heavy chains (mhc) A and B, the body wall muscle isoforms (Venolia & Waterston, Genetics 126:345, 1990). We have found that the ts mutation
e286 does not lead to alterations in the accumulation of mhc A and B in strains grown at the restrictive temperature. Analysis of filaments purified from strains grown at the restrictive temperature has shown that both mhc isoforms are incorporated into thick filaments, although their quantity is markedly decreased when compared to those of strains grown at the permissive temperature. The
unc-45 gene has been cloned and is predicted to encode a 961 amino acid residue protein (Venolia, L. pers. comm.). We have identified two domains: an amino-terminal region which contains three tandem TPR (tetratricopeptide repeat) motifs and a carboxy-terminal CRO1/SHE4 domain. TPR motifs consist of antiparallel a-helices which pack together into an amphipathic groove which mediates protein-protein interactions (Das et al. EMBO J. 1:1192, 1998). They are present in a variety of proteins, including chaperones and protein phosphatases. The CRO1/SHE4 domain displays primary sequence similarity to the fungal proteins Cro1p (Podospora anserina) and She4p (Saccharomyces cerevisiae). Both proteins were identified through genetic screens seeking mutants defective in processes involving differential segregation of molecules (Jansen et al. Cell 84:687, 1996; Berteaux-Lecellier et al. EMBO J. 1:1248, 1998). We have identified the genetic alterations of the five
unc-45 ts alleles. They consist of missense mutations that lead to amino acid substitutions, three of which fall on conserved residues throughout the CRO1/SHE4 domain. We have also determined that the lethal alleles are caused by premature chain termination mutations in codons located between the TPR and CRO1/SHE4 domains. These assignments further confirm the identification of the
unc-45 gene and protein. The above findings are consistent with our hypothesis of UNC-45 having an assemblase activity, that is, acting as a catalyst (e.g. chaperone or scaffold) during thick filament assembly. Our current work is directed toward establishing whether UNC-45 is a structural component of thick filaments or whether it is only transiently associated with them. We are also investigating the biochemical properties of thick filaments isolated from ts strains grown at the restrictive temperature. Supported by grants from the Muscular Dystrophy Association, National Science Foundation and the National Institute of General Medical Sciences.