Expression of a nonpolymerizable actin mutant in Sf9 cells.
We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.
Joel
Peteranne B
PB
Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05405-0068, USA.
Fagnant
Patricia M
PM
Trybus
Kathleen M
KM
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000378:metabolism; D017354:Point Mutation; D011108:Polymers / Q000378:metabolism*; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology / Q000235:genetics*
D000199:Actins; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D015879:Myosin Subfragments; D011108:Polymers; D020033:Protein Isoforms; D011392:Proline; D017301:Ca(2+) Mg(2+)-ATPase; D000409:Alanine