Ion-dependent polymerization differences between mammalian beta- and gamma-nonmuscle actin isoforms

Abstract

beta- and gamma-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. beta-Actin contains an Asp(1)-Asp(2)-Asp(3) and Val(10) whereas gamma-actin has a Glu(1)-Glu(2)-Glu(3) and Ile(10). Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20-25% insect actin. Surprisingly, Ca-gamma-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than beta-actin. In the Mg-form, this difference between the two is much smaller. Ca-gamma-actin depolymerizes half as fast as does beta-actin. Mixing experiments with Ca-actins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing beta-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with gamma-actin. Phosphate release during treadmilling is twice as fast with beta- as with gamma-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of gamma-actin may cause a selective energy barrier relative to beta-actin that retards the equilibration between G- and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the gamma-actin-rich cochlear hair cell stereocilium where local mm calcium concentrations may exist.

FIGURE 1.

Locations of the structural differences between β- and γ-nonmuscle actin. A, monomer view of the crystal structure of β-actin, modified from Protein Data Bank code 1HLU (22) using Swiss-PdbViewer Version 3.7. The positions of the differing residues are color-highlighted and labeled: D1, D2, D3 green; V10, pink. B, model of the actin trimer based on the filament model of Oda et al. (21), with the positions of the differing residues color-highlighted and labeled as described above. ATP is depicted as an orange stick, and Ca²⁺ or Mg²⁺ ion depicted as a red circle.

FIGURE 2.

Polymerization kinetics of actin isoforms. Polymerization of 4.8 μm actin was initiated by the addition of magnesium and potassium chloride as described under “Experimental Procedures,” and the increase in L.S. was monitored as a function of time at 25 °C. Shown are representative plots of experiments performed at least three times with three independent actin preparations.

FIGURE 3.

Polymerization of insect and γ-actin mixtures. Polymerization of 3.5 μm total actin was initiated by the addition of magnesium and potassium chloride as described under “Experimental Procedures,” the increase in L.S. was monitored as a function of time at 25 °C. The γ-actin preparation used in this work contains 20–25% insect actin. A, γ-actin preparation, mixtures of γ-actin preparation and insect actin, and pure insect actin were polymerized, numbers behind the isoforms represent the relative percentage of each actin preparation within the reaction. B, pure insect and β-actin were polymerized. Shown are representative plots of experiments performed at least three times with three independent actin preparations.

FIGURE 4.

Co-polymerization of β- and γ-actin. Polymerization of 2.4 μm or 1.2 μm total actin was initiated by the addition of magnesium and potassium chloride as described under “Experimental Procedures,” and the increase in L.S. was monitored as a function of time at 25 °C. Shown are representative plots of experiments performed four times with two independent actin preparations.

FIGURE 5.

Polymerization of β- and γ-actin mixtures. Polymerization of 3.5 μm total actin was initiated by the addition of magnesium and potassium chloride as described under “Experimental Procedures,” and the increase in L.S. was monitored as a function of time at 25 °C. Numbers behind the isoforms represent the relative percentage of each actin isoform within the polymerization reaction. Shown are representative plots of experiments performed four times with two independent actin preparations.

FIGURE 6.

Seeded polymerization of β- and γ-actin. Polymerization of 3.5 μm actin was initiated by the addition of magnesium and potassium chloride as described under “Experimental Procedures,” in the presence or absence of PAS as indicated above and the increase in L.S. was monitored as a function of time at 25 °C. Shown are representative plots of experiments performed four times with two independent actin preparations.

FIGURE 7.

P_i release associated with polymerization of Ca²⁺ nonmuscle actin isoforms. 4.8 μm Ca²⁺ β-actin (A) and γ-actin (B) were polymerized at 25 °C by the addition of salt. Filament formation was monitored by the change in light scattering and P_i release using the Enz Check Assay. The data were normalized and superimposed as described under “Experimental Procedures.” Shown are representative plots of experiments performed at least three times with three independent actin preparations.

FIGURE 8.

Polymerization kinetics of Ca²⁺versus Mg²⁺ nonmuscle actin isoforms. Polymerization of 3.5 μm actin was initiated by the addition of magnesium and potassium chloride as described under “Experimental Procedures,” and the increase in L.S. was monitored as a function of time at 25 °C. Shown are representative plots of experiments performed at least three times with three independent actin preparations.

FIGURE 9.

P_i release associated with polymerization of Mg-nonmuscle actin isoforms. 4.8 μm Mg²⁺ β-actin (A) and γ-actin (B) were polymerized at 25 °C by the addition of salt. Filament formation was monitored by the change in light scattering and P_i release using the Enz Check Assay. The data were normalized and were superimposed as described under “Experimental Procedures.” Shown are representative plots of experiments performed at least three times with three independent actin preparations.