Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms

Kuo-Kuang Wen,Peter A Rubenstein

doi:10.1074/jbc.m109.006981

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.

Highlights

It has been shown that this complex modifies the kinetics of actin dynamics at the barbed end [9, 11, 18, 22]
Interaction of Bnr1 and Bni1 with Pyrene-labeled Yeast G-actin—To begin to assess a possible difference in the ability of the two yeast formins to interact with G-actin, we examined their interaction with yeast G-actin in the Mg-form labeled at Cys-374 with pyrene maleimide
Because actin conformation is known to be affected by the nature of the cation at the high affinity cation binding site [35, 36], we examined the interaction of the formins with Ca-G-actin

Summary

Introduction

Addition of Bnr1 to Mg-G-actin in low ionic strength buffer caused a dose-dependent increase in actin pyrene fluorescence (Fig. 1A). We wished to assess the differential effects of Bni1 and Bnr1 on the salt-induced polymerization of Cys-374 pyrene-labeled yeast actin.

Results

Conclusion