Yeast actin with a subdomain 4 mutation (A204C) exhibits increased pointed-end critical concentration

Abstract

Characterizing mutants of actin that do not polymerize will advance our understanding of the mechanism of actin polymerization and will be invaluable for the production of short F-actin structures for structural studies. To circumvent the problem of expressing dominant lethal nonpolymerizing actin in yeast, we adopted a cysteine engineering strategy. Here we report the characterization of a mutant of yeast actin, AC-actin, possessing a single pointed-end mutation, A204C. Expression of this mutant in yeast results in actin-polymerization-deficient phenotypes. When copolymerized with wild-type actin, ATP-AC-actin is incorporated into filaments. ADP-AC-actin participates in the nucleation and elongation of wild-type filaments only at very high concentrations. At low concentrations, ADP-AC-actin appears to participate only in the nucleation of wild-type filaments, suggesting that Ala-204 is involved in modulating the critical concentration of the pointed end of actin.

Cysteine engineering of actin self-assembly interfaces

“…Of these positions, previous data regarding substitutions at three were available: the D286A/D288A mutant is recessive lethal in yeast (Wertman et al 1992) and mutations at Gly245 exhibit reduced polymerization (Aspenström et al 1992). We previously reported our findings with the A204C mutant (Teal and Dawson 2007), which exhibited a markedly increased Cc at the pointed end.…”

“…Yeast cell F-actin structures were visualized by fluorescence microscopy of rhodamine-phalloidin with a Nikon Eclipse E600 fluorescent microscope equipped with a CoolSnapfx monochrome CCD digital camera (Roper Scientific, Tucson, Arizona) and processed using Metamorph software (Molecular Devices, Downingtown, Pennsylvania) as described previously (Teal and Dawson 2007).…”

“…The ability of mutant actin to polymerize was analyzed by sedimentation at 361 000g or light scattering at an excitation and emission of 360 nm as described previously (Teal and Dawson 2007). The critical concentration (Cc) was determined by measuring the light scattering intensity for both G-and F-actin in a series of protein concentrations.…”

“…Most of the cysteine-engineered ACT1-expressing yeast strains possessed doubling times within the range of 2.5-3 h (Table 1), including the strain expressing the A204C mutant that has been found to exhibit a very high Cc (Teal and Dawson 2007). Both heat-sensitive D288C-and T149C-expressing strains had higher doubling times of 3.5 h.…”

“…To achieve this goal, we tested the interactions at the interfaces of F-actin contacts through a cysteine-engineering approach. Previously, we characterized a position at the pointed end of actin at residue 204 that, when mutated to cysteine, exhibited reduced polymerization characteristics (Teal and Dawson 2007). Here, we present our characterization of other mutants of actin where cysteine has been engineered at interface positions suggested by the Holmes F-actin model.…”