Mutation of actin Tyr-53 alters the conformations of the DNase I-binding loop and the nucleotide-binding cleft

Abstract

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary "preference" for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations.

FIGURE 1.

SDS-PAGE of purified actins. Typical preparations of purified endogenous WT actin and expressed Y/Y, Y/F, Y/A, and Y/E actins were analyzed on 10% polyacrylamide gels; 10 μg of proteins was added to each lane.

FIGURE 2.

Subtilisin digestion of actins. WT, Y/Y, Y/F, Y/A, and Y/E actins were incubated with subtilisin (see “Experimental Procedures”), and aliquots were taken at the indicated times for SDS-PAGE analysis on 10% polyacrylamide gels.

FIGURE 3.

Properties of monomeric actins. A, inhibition of DNase I activity as a function of actin concentration (see “Experimental Procedures”). B, time course of nucleotide exchange. Monomeric actin (3 μm) with bound etheno-ATP was incubated with 0.1 m ATP, and the decrease in fluorescence was monitored.

FIGURE 4.

Polymerization of actins. A, critical concentration; actin was polymerized overnight. B, time course of polymerization of 6 μm actin. All polymerizations were at room temperature in G-buffer plus 100 mm KCl and 2 mm MgCl₂. AU, arbitrary units.

FIGURE 5.

Electron microscopy of polymerized actins. Negatively stained images of polymerized actins are shown.

FIGURE 6.

ATP hydrolysis accompanying polymerization. A, ATP hydrolysis. B, polymerization. The time course of polymerization was followed by the increase in light scattering, and the time course of ATP hydrolysis was followed by an assay of P_i release (see “Experimental Procedures”). Actin concentrations were as follows: WT, Y/Y, and Y/F, 6 μm; Y/A and Y/E, 8 μm. AU, arbitrary units.

FIGURE 7.

Actin activation of myosin S1 MgATPase activity. A, assays conducted in the absence of phalloidin. B, assays done in the presence of phalloidin (1:1 molar ratio to actin) to stabilize the actin filaments. The kinetic values from these assays are in Table 3.

FIGURE 8.

Properties of purified Y/W and Y/L actins compared with WT actin. A, SDS-PAGE of purified actins. B, nucleotide exchange of Y/W and Y/L actins is faster than for WT. C, Y/W polymerized at the same rate as WT, but Y/L polymerized more slowly. D, the critical concentrations of Y/W, Y/L, and WT are similar. E, Y/W and Y/L form filaments indistinguishable from filaments of WT. AU, arbitrary units.

FIGURE 9.

Expression of FLAG-TEVCS-tagged mutant actins and their co-localization with endogenous actin. Cells expressing the mutant actins were grown in suspension culture and then fixed and double-stained with anti-actin antibody (red) to visualize both endogenous and mutant actin and with anti-FLAG antibody (green) to visualize only the mutant actin. DIC, differential interference contrast microscopy. Bar, 10 μm. The percentages of cells that reacted with anti-FLAG antibody, with the number of cells counted in parentheses, were as follows: Y/Y, 95% (143); Y/F, 94% (178); Y/W, 92% (104); Y/L, 83% (198); Y/A, 91% (143); Y/E, 64% (425).

FIGURE 10.

Growth of cells expressing mutant actins. A, immunoblots of SDS-PAGE of total cell proteins of cells expressing FLAG-TEVCS-mutant actins. Upper gel, anti-FLAG antibody; lower gel, anti-actin antibody. B, growth curves of WT cells and cells expressing Y/Y, Y/F, Y/A, and Y/E actin. Cells expressing Y/A grew more slowly than Y/Y cells and to a lower maximum cell density. C, growth curves of cells expressing Y/Y, Y/L, and Y/W actin. Y/L and Y/W cells grew more slowly than Y/Y cells and to a lower maximum density. Because the plots are exponential, differences in growth rates and final cell densities appear to be less than they are (see “Results”).

FIGURE 11.

Development of cells expressing mutant actins. Images of the cell lines analyzed in Fig. 9 were taken after development for 48 h. As discussed under “Results,” Y/Y, Y/F, Y/W, and Y/L cells formed mature fruiting bodies similar to those formed by WT cells except that their heads were 25% smaller. Y/A cells never developed beyond the mound stage, and only about half of the mounds of Y/E cells developed into fruiting bodies by 48 h, with heads 25% smaller than those of Y/Y cells.