Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6.
Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities and how they may be regulated differentially. We isolated and compared the activities of carboxyl-terminal fragments of the only two formins expressed in Saccharomyces cerevisiae, Bni1 and Bnr1. Bnr1 was an order of magnitude more potent than Bni1 in actin nucleation and processive capping, and unlike Bni1, Bnr1 bundled actin filaments. Profilin bound directly to Bni1 and Bnr1 and regulated their activities similarly. However, the cell polarity factor Bud6/Aip3 specifically bound to and stimulated Bni1, but not Bnr1. This was unexpected, since previous two-hybrid studies suggested Bud6 interacts with both formins. We mapped Bud6 binding activity to specific residues in the carboxyl terminus of Bni1 that are adjacent to its diaphanous autoregulatory domain (DAD). Fusion of the carboxyl terminus of Bni1 to Bnr1 conferred Bud6 stimulation to a Bnr1-Bni1 chimera. Thus, Bud6 differentially stimulates Bni1 and not Bnr1. We found that Bud6 is up-regulated during bud growth, when it is delivered to the bud tip on Bni1-nucleated actin cables. We propose that Bud6 stimulation of Bni1 promotes robust cable formation, which in turn delivers more Bud6 to the bud tip, reinforcing polarized cell growth through a positive feedback loop.
Moseley
James B
JB
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.
Goode
Bruce L
BL
D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D049109:Cell Proliferation; D003285:Contractile Proteins / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D015151:Immunoblotting; D047468:Immunoprecipitation; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051304:Profilins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation
D000199:Actins; C106463:BNR1 protein, S cerevisiae; C107727:BUD6 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D051304:Profilins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins