Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium.
Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an approximately 100-fold increase in the mutants' affinity for actin in the presence of ATP.
Tsiavaliaris
Georgios
G
Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany.
Fujita-Becker
Setsuko
S
Batra
Renu
R
Levitsky
Dmitrii I
DI
Kull
F Jon
FJ
Geeves
Michael A
MA
Manstein
Dietmar J
DJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002455:Cell Division / Q000502:physiology; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000502:physiology*; D008024:Ligands; D008958:Models, Molecular; D020409:Molecular Motor Proteins; D009154:Mutation; D024462:Myosin Type II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000199:Actins; D008024:Ligands; D020409:Molecular Motor Proteins; D024462:Myosin Type II