Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.
The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle.
Department of Cellular Physiology and Centre for Nanosciences (CeNS), Ludwig-Maximilians-Universität München, Schillerstrasse 44, 80336 München, Germany.
Christopher
C
Batters
Katy A
KA
Woodall
Christopher P
CP
Toseland
Christian
C
Hundschell
Claudia
C
Veigel
D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007891:Leishmania / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins
D002147:Calmodulin; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D009218:Myosins