Unc-45 mutations in Caenorhabditis elegans implicate a CRO1/She4p-like domain in myosin assembly

The Caenorhabditis elegans unc-45 locus has been proposed to encode a prote in machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. C RO1 and She4p are fungal proteins required for the segregation of other mol ecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residu es within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent t ranslation of the COOH-terminal domain. Electron microscopy shows that thic k filament accumulation in vivo is decreased by similar to 50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that a ssemble have abnormal structure. Immunofluorescence and immunoelectron micr oscopy show that myosins A and B are scrambled, in contrast to their assemb ly into distinct regions at the permissive temperature and in wild type. Th is abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortene d lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-lik e region in the assembly of myosin isoforms in C. elegans and suggest a pos sible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) pro tein family.