title: A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.
abstract: Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.
authors.affiliation: Departments of Molecular Physiology and Biophysics and University of Vermont, Burlington, Vermont 05405.
authors.forename: Carol S
authors.initials: CS
authors.lastname: Bookwalter
authors.affiliation: Departments of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405.
authors.forename: Anne
authors.initials: A
authors.lastname: Kelsen
authors.affiliation: Departments of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Leung
authors.affiliation: Departments of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405. Electronic address: gary.ward@uvm.edu.
authors.forename: Gary E
authors.initials: GE
authors.lastname: Ward
authors.affiliation: Departments of Molecular Physiology and Biophysics and University of Vermont, Burlington, Vermont 05405. Electronic address: kathleen.trybus@uvm.edu.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Trybus
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000737:chemistry; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000737:chemistry; D018995:Myosin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry*; D018994:Myosin Light Chains / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry* / Q000502:physiology; D061987:Sf9 Cells; D012995:Solubility; D018411:Spodoptera; D014122:Toxoplasma / Q000378:metabolism*
chemical_list: D000199:Actins; D018832:Molecular Chaperones; D018994:Myosin Light Chains; D015800:Protozoan Proteins; D018995:Myosin Heavy Chains; D002118:Calcium
keywords: ATPase; Class XIV Myosin; Molecular Chaperone; Molecular Motor; Myosin Chaperones; Myosin Motor Complex; Protein Expression; Protein Folding; Toxoplasma gondii; UCS Proteins