Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain (*)
- From the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611
- § To whom correspondence should be addressed. Tel.: 312-503-4151; Fax: 312-503-5994.
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 [Medline] 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 NH
-terminal 11 or 28 amino acid residues (ΔN11 or ΔN28) or the COOH-terminal 15 amino acid residues (ΔC15) 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, ΔN28 and
ΔN11 bound to the MHC at 15% of WT levels and ΔC15 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.
Footnotes
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↵* This work was supported in part by Grant GM-39264 from the National Institutes of Health (to R. L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- MHC
-
myosin heavy chain
- ELC
-
essential light chain
- RLC
-
regulatory light chain
- CaM
-
calmodulin
- WT
-
wild-type
- PCR
-
polymerase chain reaction
- PIPES
-
1,4-piperazinediethanesulfonic acid
- BSA
-
bovine serum albumin
- DAPI
-
4′,6-diamino-2-phenylindole-2HCl.
-
↵2 Chen, T.-L. L., Kowalczyk, P. A., Ho, G., and Chisholm, R. L., J. Cell Sci., in press.
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- Received January 26, 1995.
- Revision received August 21, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
