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Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the regulatory light chain
S R Tafuri1, A M Rushforth, E R Kuczmarski
1Department of Cell Biology and Anatomy, Northwestern University Medical School, Chicago, Illinois 60611.
Abstract:
Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.
Insights
Researchers characterized Dictyostelium regulatory light chains (RMLC) of nonmuscle myosin. They found the RMLC gene is single and its mRNA expression is coordinated with essential myosin light chains during development.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nonmuscle myosin regulatory light chains (RMLC) phosphorylation influences actin-activated ATPase activity and filament formation.
- The precise regulatory mechanisms and RMLC involvement in ATP hydrolysis remain largely uncharacterized.
Purpose of the Study:
- To isolate and characterize cDNAs encoding Dictyostelium nonmuscle RMLC.
- To elucidate the structural and regulatory properties of Dictyostelium RMLC.
Main Methods:
- Screening of a lambda gt11 expression library using an RMLC-specific antibody.
- Obtaining full-length cDNA through hybridization with an initial clone.
- Amino acid sequence prediction and comparison with known proteins.
- Genomic Southern blot analysis.
- Analysis of RMLC mRNA expression patterns during Dictyostelium development.
Main Results:
- A composite RMLC cDNA of 645 nucleotides was obtained, predicting an 18,300-dalton protein.
- The predicted amino acid sequence showed 42% identity to Dictyostelium calmodulin and 30% to chicken skeletal myosin RMLC.
- Identified E-F hand calcium-binding-like domains and a phosphorylation sequence similar to chicken myosin light chains.
- Genomic Southern blot indicated a single RMLC gene in Dictyostelium.
- RMLC mRNA accumulation increased before aggregation and during culmination, mirroring essential myosin light chain expression.
Conclusions:
- The Dictyostelium RMLC shares structural similarities with calmodulin and other myosin light chains, including calcium-binding and phosphorylation sites.
- The Dictyostelium genome contains a single RMLC gene.
- Coordinated expression of RMLC and essential myosin light chain suggests synchronized regulation during Dictyostelium development.