title: 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.
authors.affiliation: From the Department of Biochemistry, Roy A. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242.
authors.forename: Kuo-Kuang
authors.initials: KK
authors.lastname: Wen
authors.affiliation: From the Department of Biochemistry, Roy A. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242. Electronic address: peter-rubenstein@uiowa.edu.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Rubenstein
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D046912:Multiprotein Complexes; D009154:Mutation; D009994:Osmolar Concentration; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000199:Actins; C106463:BNR1 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins