Differential regulation of actin polymerization and structure by yeast formin isoforms

Abstract

The budding yeast formins, Bnr1 and Bni1, behave very differently with respect to their interactions with muscle actin. However, the mechanisms underlying these differences are unclear, and these formins do not interact with muscle actin in vivo. We use yeast wild type and mutant actins to further assess these differences between Bnr1 and Bni1. Low ionic strength G-buffer does not promote actin polymerization. However, Bnr1, but not Bni1, causes the polymerization of pyrene-labeled Mg-G-actin in G-buffer into single filaments based on fluorometric and EM observations. Polymerization by Bnr1 does not occur with Ca-G-actin. By cosedimentation, maximum filament formation occurs at a Bnr1:actin ratio of 1:2. The interaction of Bnr1 with pyrene-labeled S265C Mg-actin yields a pyrene excimer peak, from the cross-strand interaction of pyrene probes, which only occurs in the context of F-actin. In F-buffer, Bnr1 promotes much faster yeast actin polymerization than Bni1. It also bundles the F-actin in contrast to the low ionic strength situation where only single filaments form. Thus, the differences previously observed with muscle actin are not actin isoform-specific. The binding of both formins to F-actin saturate at an equimolar ratio, but only about 30% of each formin cosediments with F-actin. Finally, addition of Bnr1 but not Bni1 to pyrene-labeled wild type and S265C Mg-F actins enhanced the pyrene- and pyrene-excimer fluorescence, respectively, suggesting Bnr1 also alters F-actin structure. These differences may facilitate the ability of Bnr1 to form the actin cables needed for polarized delivery of nutrients and organelles to the growing yeast bud.

FIGURE 1.

Interaction of Bnr1 with pyrene-labeled Mg-G-actin. Panel A, aliquots of 0.5 μm pyrene-labeled Mg-G-actin were combined with increasing amounts of Bnr1 (line 1: 0 μm, line 2: 0.1 μm, and line 3: 0.5 μm) in Mg-G-buffer conditions (10 mm Tris-HCl pH 7.5, 0.2 mm MgCl₂, 0.2 mm ATP, 100 μm EGTA, and 1 mm DTT), and the solutions were incubated at room temperature for 15 min at room temperature. The fluorescence emission spectra were recorded from 375 to 500 nm following excitation at 365 nm. Panel B, pyrene-labeled Mg G-actin was combined with Bnr1 (Δ: no Bnr1, and ○: 0.5 μm) in Mg-G-buffer, and the increase in pyrene fluorescence intensity at emission wavelength 385 nm was recorded over time. Excitation wavelength was 365 nm. Panel C, net change pyrene fluorescence intensity at emission wavelength 385 nm from panel A was plotted against the concentration of Bnr1 as described under “Experimental Procedures.” The experiments shown in each panel have been repeated twice with essentially the same results.

FIGURE 2.

Electron microscopic analysis of the structure and length of the Bnr1/G-actin complex. Panel A, either 1 μm Ca- or Mg-G-actin was mixed with 1 μm Bnr1 in either Ca- or Mg-G-buffer, respectively, at room temperature for 15 min, and the samples from each reaction were observed by EM as described under “Experimental Procedures.” Bar = 0.2 μm. Panel B, distribution of filament length in the reaction of 1 μm Mg-G-actin with Bnr1 (solid column: 0.1 μm, and open column: 1 μm) were determined by measuring at least 100 filaments per sample by ImageJ (NIH), and the distribution of filament length was analyzed with Excel (Microsoft).

FIGURE 3.

Interaction of pyrene S265C G-actin with Bnr1. Panel A, pyreneS265C Mg-G-actin, 1 μm, was mixed with Bnr1 (line 1: no formin and line 2: 1 μm) in Mg- G-buffer at room temperature for 15 min, and the fluorescence emission spectra were recorded from 370–600 nm following excitation at 365 nm. Panel B, normalized net change in fluorescence at 485 nm from the titration was plotted against the concentration of Bnr1 in each reaction. The data are averaged from three individual experiments.

FIGURE 4.

Cosedimentation analysis of the Bnr1/G-actin interaction. Panel A, Mg-G-actin, 1 μm, was mixed with different concentrations of Bnr1 (as indicated on the top of the gels) G-buffer conditions at room temperature for 20 min, and the mixture was then centrifuged. The entire pellet fraction (P) and one-fourth of the supernatant fraction (SN) were analyzed by 10% SDS-PAGE and Coomassie Blue staining. Details of the protocols are described under “Experimental Procedures.” Panel B, intensity of the actin and Bnr1 bands in the pellet fractions of each individual experiment were quantified by densitometry and corrected for the loading differences. The molar ratio of formin to actin in the pellet was calculated based on the intensity of each bands, corrected for differences in MW of the proteins, and plotted on the y axis against the Bnr1 concentration as described under “Experimental Procedures.” The data are averaged from four individual experiments.

FIGURE 5.

Copolymerization of G-actin with either Bnr1orBni1. Mg-G-actin (5% pyrene-labeled), 1 μm, was mixed with either Bnr1 (panel A) or Bni1 (panel B) for 15 min and was then induced to polymerize by the addition of MgCl₂ and KCl to final concentrations of 2 mm and 50 mm, respectively. The increase in pyrene fluorescence over time was recorded as described under “Experimental Procedures.” These experiments have been repeated three times with essentially identical results, and only one data set is presented. Panel A, Bnr1 concentrations are no formin (○), 3.3 μm (Õ), 10 μm (▵), 50 μm (♢), and 150 μm (□). Panel B, Bnr1 concentrations are no formin (○), 10 μm (▵), 50 μm (♢), and 150 μm (□). Arrow, time of addition of salt.

FIGURE 6.

Cosedimentation assay for the binding of yeast formins to Mg-F-actin. 1 μm Mg-actin was copolymerized in Mg-G-buffer at room temperature for 30 min with yeast formin at different concentrations as indicated on the top of the gel, and then the mixture was centrifuged. The pellet (P) and supernatant (SN) were analyzed by 10% SDS-PAGE and Coomassie Blue staining as described as under “Experimental Procedures.” Panel B, the intensity of each actin and formin band in the pellet was quantified by densitometry. The molar ratio of formin to actin in the pellet was calculated based on the intensity of each protein band, corrected for their differences in MW, and plotted on the y axis against the Bnr1 concentration as described under “Experimental Procedures.” The Bnr1 binding data are averaged from three individual experiments, and Bni1 binding data are from two individual experiments.

FIGURE 7.

EM analysis of Mg-F-actin mixed with either Bnr1 or Bni1. 1 μm Mg-actin was polymerized in the presence of 0.25 μm of either Bnr1 (panels A and B) or Bni1 (panel C) at room temperature for 30 min. The product from each reaction was examined by EM as described under “Experimental Procedures.” Panel B, enlarged Bnr1/F actin bundle compared with the bundle in panel A. Bar = 0.2 μm.

FIGURE 8.

Pyrene-excimer resulting from the copolymerization of pyrene-S265C Mg-actin and Bnr1 in F-salts. Panel A, 1 μm pyreneS265C Mg-actin was copolymerized with Bnr1 (line 1: no formin and line 2: 0. 5 μm) following induction of polymerization with MgCl₂ and KCl, and the fluorescence emission spectrum of this reaction was recorded from 375 to 600 nm using an excitation wavelength of 365 nm. Panel B, normalized net change in fluorescence intensity at 485 nm was plotted against the concentration of Bnr1 as described under “Experimental Procedures.” These experiments have been repeated twice with essentially same results, and the data presented here are the average of these two experiments.