Abstract
Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and Chisholm, R. L. (1992) Cell 69, 951-962). Since normal myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the NH2-terminal 11 or 28 amino acid residues (delta N11 or delta N28) or the COOH-terminal 15 amino acid residues (delta C15) abolished binding of the ELC to the MHC when the mutants were expressed in wild-type (WT) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less efficient competition with WT ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE-) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, delta N28 and delta N11 bound to the MHC at 15% of WT levels and delta C15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE- cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo.
Highlights
Sequence comparisons of essential light chain (ELC) from various sources show that the COOH-terminal half of the molecule is more conserved than the NH2-terminal half [5, 6] and that it may represent the site required for association with myosin heavy chain (MHC)
The myc Epitope-tagged WT ELC Associates with the MHC—To facilitate the detection of ELC mutants when expressed in WT cells, the ELCs were tagged at the carboxyl termini with a myc epitope [27] of 10 amino acid residues (EQKLISEEDL) and expressed in Dictyostelium AX3 cells using an expression vector pBORP [21]
The structure of CaM1⁄7M13 peptide complex shows that residues in both lobes of calmodulin are in contact with the M13 peptide (a 26-amino acid peptide containing the CaM binding domain of myosin light chain kinase); residues in the helices account for the vast majority of those contacts [29, 36]
Summary
Sequence comparisons of ELCs from various sources show that the COOH-terminal half of the molecule is more conserved than the NH2-terminal half [5, 6] and that it may represent the site required for association with MHC. The atomic structures of calmodulin (CaM) and troponin C show two terminal globular lobes linked by a central helix, with each lobe containing a pair of EF-hands [10, 11]. Threedimensional structures of myosin S1 [13] and the regulatory domain of scallop myosin [14] confirmed these predictions Based on these structures, we divided the Dictyostelium ELC into three structural domains: an NH2-terminal globular domain, a COOH globular domain, and a central linker domain. We divided the Dictyostelium ELC into three structural domains: an NH2-terminal globular domain, a COOH globular domain, and a central linker domain The participation of these domains in ELC-MHC interaction was assessed. The ability of mutant ELC to associate with the MHC in vivo and to rescue the phenotypic defects of mlcEϪ cells was analyzed
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