• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of geneticsGeneticsCurrent IssueInformation for AuthorsEditorial BoardSubscribeSubmit a Manuscript
Genetics. Sep 1998; 150(1): 43–58.
PMCID: PMC1460329

Identification of functional connections between calmodulin and the yeast actin cytoskeleton.

Abstract

One of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, cmd1A, results in a characteristic functional defect in actin organization. We report here that among the complementing mutations, a representative cmd1A mutation (cmd1-226: F92A) is synthetically lethal with a mutation in MYO2 that encodes a class V unconventional myosin with calmodulin-binding domains. Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein. Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggesting an importance of hydrophobic interaction between calmodulin and Myo2p. To analyze other components involved in actin organization through calmodulin, we isolated and characterized mutations that show synthetic lethal interaction with cmd1-226; these "cax" mutants fell into five complementation groups. Interestingly, all the mutations themselves affect actin organization. Unlike cax2, cax3, cax4, and cax5 mutations, cax1 shows allele-specific synthetic lethality with the cmd1A allele. CAX1 is identical to ANP1/GEM3/MCD2, which is involved in protein glycosylation. CAX4 is identical to the ORF YGR036c, and CAX5 is identical to MNN10/SLC2/BED1. We discuss possible roles for Cax proteins in the regulation of the actin cytoskeleton.

Full Text

The Full Text of this article is available as a PDF (413K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Johnston GC, Prendergast JA, Singer RA. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. [PMC free article] [PubMed]
  • Jones JS, Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. [PubMed]
  • Babu YS, Bugg CE, Cook WJ. Structure of calmodulin refined at 2.2 A resolution. J Mol Biol. 1988 Nov 5;204(1):191–204. [PubMed]
  • Ballou L, Hitzeman RA, Lewis MS, Ballou CE. Vanadate-resistant yeast mutants are defective in protein glycosylation. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3209–3212. [PMC free article] [PubMed]
  • Karpova TS, Lepetit MM, Cooper JA. Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth. Genetics. 1993 Nov;135(3):693–709. [PMC free article] [PubMed]
  • Brockerhoff SE, Stevens RC, Davis TN. The unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae. J Cell Biol. 1994 Feb;124(3):315–323. [PMC free article] [PubMed]
  • Kilmartin JV, Dyos SL, Kershaw D, Finch JT. A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated. J Cell Biol. 1993 Dec;123(5):1175–1184. [PMC free article] [PubMed]
  • Chapman RE, Munro S. The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway. EMBO J. 1994 Oct 17;13(20):4896–4907. [PMC free article] [PubMed]
  • Koshland D, Kent JC, Hartwell LH. Genetic analysis of the mitotic transmission of minichromosomes. Cell. 1985 Feb;40(2):393–403. [PubMed]
  • Cheney RE, O'Shea MK, Heuser JE, Coelho MV, Wolenski JS, Espreafico EM, Forscher P, Larson RE, Mooseker MS. Brain myosin-V is a two-headed unconventional myosin with motor activity. Cell. 1993 Oct 8;75(1):13–23. [PubMed]
  • Lillie SH, Brown SS. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae. J Cell Biol. 1994 May;125(4):825–842. [PMC free article] [PubMed]
  • McKnight GL, Cardillo TS, Sherman F. An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell. 1981 Aug;25(2):409–419. [PubMed]
  • Davis TN, Thorner J. Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7909–7913. [PMC free article] [PubMed]
  • Mercer JA, Seperack PK, Strobel MC, Copeland NG, Jenkins NA. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature. 1991 Feb 21;349(6311):709–713. [PubMed]
  • Mermall V, Miller KG. The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm. J Cell Biol. 1995 Jun;129(6):1575–1588. [PMC free article] [PubMed]
  • Espindola FS, Espreafico EM, Coelho MV, Martins AR, Costa FR, Mooseker MS, Larson RE. Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin. J Cell Biol. 1992 Jul;118(2):359–368. [PMC free article] [PubMed]
  • Mondésert G, Clarke DJ, Reed SI. Identification of genes controlling growth polarity in the budding yeast Saccharomyces cerevisiae: a possible role of N-glycosylation and involvement of the exocyst complex. Genetics. 1997 Oct;147(2):421–434. [PMC free article] [PubMed]
  • Geli MI, Riezman H. Role of type I myosins in receptor-mediated endocytosis in yeast. Science. 1996 Apr 26;272(5261):533–535. [PubMed]
  • Mulholland J, Wesp A, Riezman H, Botstein D. Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle. Mol Biol Cell. 1997 Aug;8(8):1481–1499. [PMC free article] [PubMed]
  • Novick P, Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. [PubMed]
  • Geiser JR, Sundberg HA, Chang BH, Muller EG, Davis TN. The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Dec;13(12):7913–7924. [PMC free article] [PubMed]
  • Ohya Y, Anraku Y. Yeast calmodulin: structural and functional elements essential for the cell cycle. Cell Calcium. 1992 Jun-Jul;13(6-7):445–455. [PubMed]
  • Goodson HV, Anderson BL, Warrick HM, Pon LA, Spudich JA. Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton. J Cell Biol. 1996 Jun;133(6):1277–1291. [PMC free article] [PubMed]
  • Ohya Y, Botstein D. Diverse essential functions revealed by complementing yeast calmodulin mutants. Science. 1994 Feb 18;263(5149):963–966. [PubMed]
  • Govindan B, Bowser R, Novick P. The role of Myo2, a yeast class V myosin, in vesicular transport. J Cell Biol. 1995 Mar;128(6):1055–1068. [PMC free article] [PubMed]
  • Ohya Y, Botstein D. Structure-based systematic isolation of conditional-lethal mutations in the single yeast calmodulin gene. Genetics. 1994 Dec;138(4):1041–1054. [PMC free article] [PubMed]
  • Haarer BK, Petzold A, Lillie SH, Brown SS. Identification of MYO4, a second class V myosin gene in yeast. J Cell Sci. 1994 Apr;107(Pt 4):1055–1064. [PubMed]
  • Ohya Y, Uno I, Ishikawa T, Anraku Y. Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae. Eur J Biochem. 1987 Oct 1;168(1):13–19. [PubMed]
  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. [PubMed]
  • Palzkill T, Botstein D. Probing beta-lactamase structure and function using random replacement mutagenesis. Proteins. 1992 Sep;14(1):29–44. [PubMed]
  • Palzkill T, Botstein D. Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase. J Bacteriol. 1992 Aug;174(16):5237–5243. [PMC free article] [PubMed]
  • Paravicini G, Cooper M, Friedli L, Smith DJ, Carpentier JL, Klig LS, Payton MA. The osmotic integrity of the yeast cell requires a functional PKC1 gene product. Mol Cell Biol. 1992 Nov;12(11):4896–4905. [PMC free article] [PubMed]
  • Sobue K, Muramoto Y, Fujita M, Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5652–5655. [PMC free article] [PubMed]
  • Piazza GA, Wallace RW. Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1683–1687. [PMC free article] [PubMed]
  • Stirling DA, Welch KA, Stark MJ. Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body. EMBO J. 1994 Sep 15;13(18):4329–4342. [PMC free article] [PubMed]
  • Pringle JR, Preston RA, Adams AE, Stearns T, Drubin DG, Haarer BK, Jones EW. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. [PubMed]
  • Takagishi Y, Oda S, Hayasaka S, Dekker-Ohno K, Shikata T, Inouye M, Yamamura H. The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice. Neurosci Lett. 1996 Sep 13;215(3):169–172. [PubMed]
  • Rasmussen CD, Means RL, Lu KP, May GS, Means AR. Characterization and expression of the unique calmodulin gene of Aspergillus nidulans. J Biol Chem. 1990 Aug 15;265(23):13767–13775. [PubMed]
  • Takahashi K, Tago K, Okano H, Ohya Y, Katada T, Kanaho Y. Augmentation by calmodulin of ADP-ribosylation factor-stimulated phospholipase D activity in permeabilized rabbit peritoneal neutrophils. J Immunol. 1996 Feb 1;156(3):1229–1234. [PubMed]
  • Takeda T, Yamamoto M. Analysis and in vivo disruption of the gene coding for calmodulin in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3580–3584. [PMC free article] [PubMed]
  • Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. [PubMed]
  • Yasui K, Kitamoto K, Gomi K, Kumagai C, Ohya Y, Tamura G. Cloning and nucleotide sequence of the calmodulin-encoding gene (cmdA) from Aspergillus oryzae. Biosci Biotechnol Biochem. 1995 Aug;59(8):1444–1449. [PubMed]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PMC free article] [PubMed]

Articles from Genetics are provided here courtesy of Genetics Society of America

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...