A Novel System for Expressing Toxic Actin Mutants in Dictyostelium and Purification and Characterization of a Dominant Lethal Yeast Actin Mutant

Taro Q.P Noguchi,Noriko Kanzaki,Hironori Ueno,Keiko Hirose,Taro Q.P Uyeda

doi:10.1074/jbc.m703165200

Abstract

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.

Highlights

Able to take different conformations and that, in certain cases, large blocks of subunits spanning long distances within individual filaments undergo cooperative conformational changes [1,2,3,4,5,6]
Actin fused N-terminally to GFP through a flexible linker is able to copolymerize normally with endogenous actin in Dictyostelium [19, 27], and we were able to evaluate the expression of the mutant form separately from the endogenous actin by SDS-PAGE
We have developed a novel system for expressing recombinant actin in which His-tagged thymosin

Summary

Introduction

Able to take different conformations and that, in certain cases, large blocks of subunits spanning long distances within individual filaments undergo cooperative conformational changes [1,2,3,4,5,6]. If incorporated into an actin filament the triple mutation would be near the pointed end surface of the molecule, while the double mutation would be exposed on the barbed end surface This suggests the possibility that these two mutants modify the properties of either end of the filaments in the cells, thereby interfering with the functions of the wild-type molecules within the same cells [8]. An and Mogami [9] took advantage of these unique features of Act88F and identified 15 dominant negative actin alleles that resulted in a no flight phenotype in the presence of a wild-type allele. Aspenstrom and Karlsson [12] and Sutoh et al [13] were the first to express a recombinant actin mutant, using S. cerevisiae and the cellular slime mold D. discoideum, respectively In these cases, it was possible to separate the mutant and endogenous actins using ion exchange chromatography. This is promising, but with this system it would be difficult to separate the mutant from the endogenous actin if the actin mutant was polymerization-competent, and it has not been shown to express dominant negative mutant actins with reasonable yields

Methods

Results

Conclusion