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. 1997 Sep 15;16(18):5520–5530. doi: 10.1093/emboj/16.18.5520

Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis.

P Lappalainen 1, E V Fedorov 1, A A Fedorov 1, S C Almo 1, D G Drubin 1
PMCID: PMC1170184  PMID: 9312011

Abstract

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive-lethal and three were conditional-lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F-actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment-1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N-terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.

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Selected References

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  1. Abe H., Obinata T., Minamide L. S., Bamburg J. R. Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development. J Cell Biol. 1996 Mar;132(5):871–885. doi: 10.1083/jcb.132.5.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bass S. H., Mulkerrin M. G., Wells J. A. A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4498–4502. doi: 10.1073/pnas.88.10.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  4. Gunsalus K. C., Bonaccorsi S., Williams E., Verni F., Gatti M., Goldberg M. L. Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis. J Cell Biol. 1995 Dec;131(5):1243–1259. doi: 10.1083/jcb.131.5.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hatanaka H., Ogura K., Moriyama K., Ichikawa S., Yahara I., Inagaki F. Tertiary structure of destrin and structural similarity between two actin-regulating protein families. Cell. 1996 Jun 28;85(7):1047–1055. doi: 10.1016/s0092-8674(00)81305-7. [DOI] [PubMed] [Google Scholar]
  6. Hawkins M., Pope B., Maciver S. K., Weeds A. G. Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments. Biochemistry. 1993 Sep 28;32(38):9985–9993. doi: 10.1021/bi00089a014. [DOI] [PubMed] [Google Scholar]
  7. Hayden S. M., Miller P. S., Brauweiler A., Bamburg J. R. Analysis of the interactions of actin depolymerizing factor with G- and F-actin. Biochemistry. 1993 Sep 28;32(38):9994–10004. doi: 10.1021/bi00089a015. [DOI] [PubMed] [Google Scholar]
  8. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Iida K., Moriyama K., Matsumoto S., Kawasaki H., Nishida E., Yahara I. Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein. Gene. 1993 Feb 14;124(1):115–120. doi: 10.1016/0378-1119(93)90770-4. [DOI] [PubMed] [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Janin J., Miller S., Chothia C. Surface, subunit interfaces and interior of oligomeric proteins. J Mol Biol. 1988 Nov 5;204(1):155–164. doi: 10.1016/0022-2836(88)90606-7. [DOI] [PubMed] [Google Scholar]
  12. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  13. Kassir Y., Simchen G. Monitoring meiosis and sporulation in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:94–110. doi: 10.1016/0076-6879(91)94009-2. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lappalainen P., Drubin D. G. Cofilin promotes rapid actin filament turnover in vivo. Nature. 1997 Jul 3;388(6637):78–82. doi: 10.1038/40418. [DOI] [PubMed] [Google Scholar]
  16. Leonard S. A., Gittis A. G., Petrella E. C., Pollard T. D., Lattman E. E. Crystal structure of the actin-binding protein actophorin from Acanthamoeba. Nat Struct Biol. 1997 May;4(5):369–373. doi: 10.1038/nsb0597-369. [DOI] [PubMed] [Google Scholar]
  17. McKim K. S., Matheson C., Marra M. A., Wakarchuk M. F., Baillie D. L. The Caenorhabditis elegans unc-60 gene encodes proteins homologous to a family of actin-binding proteins. Mol Gen Genet. 1994 Feb;242(3):346–357. doi: 10.1007/BF00280425. [DOI] [PubMed] [Google Scholar]
  18. McLaughlin P. J., Gooch J. T., Mannherz H. G., Weeds A. G. Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature. 1993 Aug 19;364(6439):685–692. doi: 10.1038/364685a0. [DOI] [PubMed] [Google Scholar]
  19. Moon A., Drubin D. G. The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. Mol Biol Cell. 1995 Nov;6(11):1423–1431. doi: 10.1091/mbc.6.11.1423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morgan T. E., Lockerbie R. O., Minamide L. S., Browning M. D., Bamburg J. R. Isolation and characterization of a regulated form of actin depolymerizing factor. J Cell Biol. 1993 Aug;122(3):623–633. doi: 10.1083/jcb.122.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ogawa K., Tashima M., Yumoto Y., Okuda T., Sawada H., Okuma M., Maruyama Y. Coding sequence of human placenta cofilin cDNA. Nucleic Acids Res. 1990 Dec 11;18(23):7169–7169. doi: 10.1093/nar/18.23.7169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reijo R. A., Cooper E. M., Beagle G. J., Huffaker T. C. Systematic mutational analysis of the yeast beta-tubulin gene. Mol Biol Cell. 1994 Jan;5(1):29–43. doi: 10.1091/mbc.5.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rosenblatt J., Agnew B. J., Abe H., Bamburg J. R., Mitchison T. J. Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails. J Cell Biol. 1997 Mar 24;136(6):1323–1332. doi: 10.1083/jcb.136.6.1323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Safer D. An electrophoretic procedure for detecting proteins that bind actin monomers. Anal Biochem. 1989 Apr;178(1):32–37. doi: 10.1016/0003-2697(89)90351-5. [DOI] [PubMed] [Google Scholar]
  25. Saito T., Lamy F., Roger P. P., Lecocq R., Dumont J. E. Characterization and identification as cofilin and destrin of two thyrotropin- and phorbol ester-regulated phosphoproteins in thyroid cells. Exp Cell Res. 1994 May;212(1):49–61. doi: 10.1006/excr.1994.1117. [DOI] [PubMed] [Google Scholar]
  26. Samstag Y., Dreizler E. M., Ambach A., Sczakiel G., Meuer S. C. Inhibition of constitutive serine phosphatase activity in T lymphoma cells results in phosphorylation of pp19/cofilin and induces apoptosis. J Immunol. 1996 Jun 1;156(11):4167–4173. [PubMed] [Google Scholar]
  27. Sutoh K., Mabuchi I. End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin. Biochemistry. 1989 Jan 10;28(1):102–106. doi: 10.1021/bi00427a015. [DOI] [PubMed] [Google Scholar]
  28. Theriot J. A., Mitchison T. J. Actin microfilament dynamics in locomoting cells. Nature. 1991 Jul 11;352(6331):126–131. doi: 10.1038/352126a0. [DOI] [PubMed] [Google Scholar]
  29. Theriot J. A., Mitchison T. J., Tilney L. G., Portnoy D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature. 1992 May 21;357(6375):257–260. doi: 10.1038/357257a0. [DOI] [PubMed] [Google Scholar]
  30. Weeds A., Maciver S. F-actin capping proteins. Curr Opin Cell Biol. 1993 Feb;5(1):63–69. doi: 10.1016/s0955-0674(05)80009-2. [DOI] [PubMed] [Google Scholar]
  31. Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yonezawa N., Homma Y., Yahara I., Sakai H., Nishida E. A short sequence responsible for both phosphoinositide binding and actin binding activities of cofilin. J Biol Chem. 1991 Sep 15;266(26):17218–17221. [PubMed] [Google Scholar]
  33. Yonezawa N., Nishida E., Koyasu S., Maekawa S., Ohta Y., Yahara I., Sakai H. Distribution among tissues and intracellular localization of cofilin, a 21kDa actin-binding protein. Cell Struct Funct. 1987 Oct;12(5):443–452. doi: 10.1247/csf.12.443. [DOI] [PubMed] [Google Scholar]
  34. Yonezawa N., Nishida E., Ohba M., Seki M., Kumagai H., Sakai H. An actin-interacting heptapeptide in the cofilin sequence. Eur J Biochem. 1989 Jul 15;183(1):235–238. doi: 10.1111/j.1432-1033.1989.tb14918.x. [DOI] [PubMed] [Google Scholar]
  35. Yonezawa N., Nishida E., Sakai H. pH control of actin polymerization by cofilin. J Biol Chem. 1985 Nov 25;260(27):14410–14412. [PubMed] [Google Scholar]

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