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. 1993 Dec 1;123(5):1185–1195. doi: 10.1083/jcb.123.5.1185

Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect

PMCID: PMC2119884  PMID: 8245125

Abstract

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F- actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin's ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.

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Selected References

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  1. Baldwin R. L. Temperature dependence of the hydrophobic interaction in protein folding. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8069–8072. doi: 10.1073/pnas.83.21.8069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  3. Bertazzon A., Tian G. H., Lamblin A., Tsong T. Y. Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium. Biochemistry. 1990 Jan 9;29(1):291–298. doi: 10.1021/bi00453a040. [DOI] [PubMed] [Google Scholar]
  4. Bremer A., Aebi U. The structure of the F-actin filament and the actin molecule. Curr Opin Cell Biol. 1992 Feb;4(1):20–26. doi: 10.1016/0955-0674(92)90054-g. [DOI] [PubMed] [Google Scholar]
  5. Bremer A., Millonig R. C., Sütterlin R., Engel A., Pollard T. D., Aebi U. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model. J Cell Biol. 1991 Nov;115(3):689–703. doi: 10.1083/jcb.115.3.689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chowdhury S., Smith K. W., Gustin M. C. Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation. J Cell Biol. 1992 Aug;118(3):561–571. doi: 10.1083/jcb.118.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cook R. K., Blake W. T., Rubenstein P. A. Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo. J Biol Chem. 1992 May 5;267(13):9430–9436. [PubMed] [Google Scholar]
  8. Cook R. K., Root D., Miller C., Reisler E., Rubenstein P. A. Enhanced stimulation of myosin subfragment 1 ATPase activity by addition of negatively charged residues to the yeast actin NH2 terminus. J Biol Chem. 1993 Feb 5;268(4):2410–2415. [PubMed] [Google Scholar]
  9. Cook R. K., Sheff D. R., Rubenstein P. A. Unusual metabolism of the yeast actin amino terminus. J Biol Chem. 1991 Sep 5;266(25):16825–16833. [PubMed] [Google Scholar]
  10. Elzinga M., Collins J. H., Kuehl W. M., Adelstein R. S. Complete amino-acid sequence of actin of rabbit skeletal muscle. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2687–2691. doi: 10.1073/pnas.70.9.2687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frieden C. The Mg2+-induced conformational change in rabbit skeletal muscle G-actin. J Biol Chem. 1982 Mar 25;257(6):2882–2886. [PubMed] [Google Scholar]
  12. Gallwitz D., Sures I. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 May;77(5):2546–2550. doi: 10.1073/pnas.77.5.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haarer B. K., Lillie S. H., Adams A. E., Magdolen V., Bandlow W., Brown S. S. Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells. J Cell Biol. 1990 Jan;110(1):105–114. doi: 10.1083/jcb.110.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  16. Milligan R. A., Whittaker M., Safer D. Molecular structure of F-actin and location of surface binding sites. Nature. 1990 Nov 15;348(6298):217–221. doi: 10.1038/348217a0. [DOI] [PubMed] [Google Scholar]
  17. Mockrin S. C., Korn E. D. Acanthamoeba profilin interacts with G-actin to increase the rate of exchange of actin-bound adenosine 5'-triphosphate. Biochemistry. 1980 Nov 11;19(23):5359–5362. doi: 10.1021/bi00564a033. [DOI] [PubMed] [Google Scholar]
  18. Ng R., Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. doi: 10.1073/pnas.77.7.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Enzymol. 1982;85(Pt B):164–181. doi: 10.1016/0076-6879(82)85020-9. [DOI] [PubMed] [Google Scholar]
  20. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  21. Privalov P. L., Gill S. J. Stability of protein structure and hydrophobic interaction. Adv Protein Chem. 1988;39:191–234. doi: 10.1016/s0065-3233(08)60377-0. [DOI] [PubMed] [Google Scholar]
  22. Schutt C. E., Lindberg U., Myslik J., Strauss N. Molecular packing in profilin: actin crystals and its implications. J Mol Biol. 1989 Oct 20;209(4):735–746. doi: 10.1016/0022-2836(89)90603-7. [DOI] [PubMed] [Google Scholar]
  23. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  24. Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
  25. Stossel T. P., Chaponnier C., Ezzell R. M., Hartwig J. H., Janmey P. A., Kwiatkowski D. J., Lind S. E., Smith D. B., Southwick F. S., Yin H. L. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  26. Strzelecka-Gołaszewska H., Venyaminov SYu, Zmorzynski S., Mossakowska M. Effects of various amino acid replacements on the conformational stability of G-actin. Eur J Biochem. 1985 Mar 1;147(2):331–342. doi: 10.1111/j.1432-1033.1985.tb08754.x. [DOI] [PubMed] [Google Scholar]
  27. Tobacman L. S., Korn E. D. The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin. J Biol Chem. 1982 Apr 25;257(8):4166–4170. [PubMed] [Google Scholar]
  28. Weeds A. G., Taylor R. S. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature. 1975 Sep 4;257(5521):54–56. doi: 10.1038/257054a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Zimmerle C. T., Frieden C. Effect of pH on the mechanism of actin polymerization. Biochemistry. 1988 Oct 4;27(20):7766–7772. doi: 10.1021/bi00420a027. [DOI] [PubMed] [Google Scholar]

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