Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2–4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants (K´d) of 52 μM for Ca2+ and 450 μM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG–MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains.
- cell motility
- Dictyostelium discoideum
- myosin light chain
The nucleotide sequence data reported has been deposited in the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AY280458.
Abbreviations used: CTER, calmodulin, troponin C, myosin II ELC and RLC; ECL®, enhanced chemiluminescence; ELC, essential light chain; ESI, electrospray ionization; GST, glutathione S-transferase; MICLC, myosin IC light chain; MlcD, myosin light chain D; RLC, regulatory light chain.
- The Biochemical Society, London ©2003