A novel system for expressing toxic actin mutants in Dictyostelium and purification and characterization of a dominant lethal yeast actin mutant.
We have developed a novel system for expressing recombinant actin in Dictyostelium. In this system, the C terminus of actin is fused to thymosin beta via a glycine-based linker. The fusion protein is purified using a His tag attached to the thymosin beta moiety and then cleaved by chymotrypsin immediately after the native final residue of actin to yield intact actin. Wild-type actin prepared in this way was functionally normal in terms of its polymerization kinetics and muscle myosin-mediated motility. We expected that this system would be particularly useful for expressing toxic actin mutants, because the actin moiety of the fusion protein is unlikely to interact with the actin cytoskeleton of the host cells. We therefore chose to express the E206A/R207A/E208A mutant, which appears to be dominant lethal in yeast, as a model case of a toxic actin mutant that is difficult to express. We found that the E206A/R207A/E208A mutant could be expressed and purified with a yield comparable to the wild-type molecule (3-4 mg/20 g cells), even though green fluorescent protein-fused actin carrying the E206A/R207A/E208A mutation was expressed at a much lower level than wild-type actin. Purified E206A/R207A/E208A actin did not polymerize, even in the presence of muscle actin; however, it accelerated polymerization of muscle actin and inhibited the nucleating and severing activities of gelsolin. Given that the location of the substituted residues is near the pointed end face of the mutant, we suggest that E206A/R207A/E208A actin behaves like a weak pointed end-capping protein that perturbs the actin cytoskeleton of the host cells.
Noguchi
Taro Q P
TQ
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology Tsukuba Central 4, 1-1-1 Highashi, Tsukuba, Ibaraki 305-8562, Japan.
Kanzaki
Noriko
N
Ueno
Hironori
H
Hirose
Keiko
K
Uyeda
Taro Q P
TQ
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000818:Animals; D001671:Biochemistry / Q000379:methods; D004023:Dictyostelium / Q000378:metabolism* / Q000502:physiology*; D018260:Gelsolin / Q000378:metabolism; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
D000199:Actins; D018260:Gelsolin; D011108:Polymers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins