Fission yeast profilin is tailored to facilitate actin assembly by the cytokinesis formin Cdc12.
The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast. We subsequently engineered ScPFY(9-Mut), a variant containing nine substitutions in the actin-binding region, which complements cdc3-124 cells. ScPFY(9-Mut), but not WT ScPFY, suppresses severe cytokinesis defects in cdc3-124 cells. Furthermore, the major activity rescued by ScPFY(9-Mut) is the ability to enhance cytokinesis formin Cdc12-mediated actin assembly in vitro, which allows cells to assemble functional contractile rings. Therefore an essential role of profilin is to specifically facilitate formin-mediated actin assembly for cytokinesis in fission yeast.
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.
Andrew J
AJ
Bestul
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.
Jenna R
JR
Christensen
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.
Agnieszka P
AP
Grzegorzewska
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.
Thomas A
TA
Burke
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.
Jennifer A
JA
Sees
Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210.
Robert T
RT
Carroll
Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210.
Vladimir
V
Sirotkin
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637.
Robert J
RJ
Keenan
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637 drkovar@uchicago.edu.
David R
DR
Kovar
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048749:Cytokinesis / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D051304:Profilins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059008:Time-Lapse Imaging / Q000379:methods
D000199:Actins; C531994:Cdc12 protein, S pombe; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D051304:Profilins; D029702:Schizosaccharomyces pombe Proteins; C490133:cdc3 protein, S pombe; D011392:Proline