, Volume 144, Issue 6, pp 665–674 | Cite as

Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1



Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.


Calcium binding EF-hand domain Heterologous expression N-terminal myristoylation Neuronal calcium sensor-1 Neurospora crassa Rattus norvegicus Site- directed mutagenesis Ultraviolet survival 



DG and RD were supported by Research Fellowships, respectively, from the Ministry of Human Resource Development (MHRD) and Council of Scientific and Industrial Research-University Grant Commission (CSIR-UGC), Government of India. We thank Prof. R. D. Burgoyne (University of Liverpool, UK) for kindly providing us the pET5α- ncs-1 Rat construct containing the R. norvegicus ncs-1 cDNA. This work was supported partially by a Grant (BT/PR3635/BCE/8/892/2012) to RT from the Department of Biotechnology, Government of India. The FGSC generously waived charges for strains and race tubes. The FGSC was supported by NSF Grant BIR-9222772.

Supplementary material

10709_2016_9933_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1194 kb)


  1. Ames JB, Ishima R, Tanaka T, Gordon JI, Stryer L, Ikura M (1997) Molecular mechanics of calcium–myristoyl switches. Nature 389:198–202  CrossRefPubMedGoogle Scholar
  2. Batistič O, Sorek N, Schültke, Yalovsky S, Kudla J (2008) Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca2+ signaling complexes in Arabidopsis. Plant Cell 20:1346–1362CrossRefPubMedPubMedCentralGoogle Scholar
  3. Benetka W, Mehlmer N, Maurer-Stroh S, Sammer M, Koranda M, Neumüller R, Betschinger J, Knoblich JA, Teige M, Eisenhaber F (2008) Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling. Cell Cycle 7:3709–3719CrossRefPubMedGoogle Scholar
  4. Bhat A, Tamuli R, Kasbekar DP (2004) Genetic transformation of Neurospora tetrasperma, demonstration of repeat-induced point mutation (RIP) in self-crosses and a screen for recessive RIP-defective mutants. Genetics 167:1155–1164CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bourne Y, Dannenberg J, Pollmann V, Marchot P, Pongs O (2001) Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1). J Biol Chem 276:11949–11955CrossRefPubMedGoogle Scholar
  6. Burgoyne RD (2007) Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat Rev Neurosci 8:182–193CrossRefPubMedPubMedCentralGoogle Scholar
  7. Burgoyne RD, Weiss JL (2001) The neuronal calcium sensor family of Ca2+-binding proteins. Biochem J 353:1–12CrossRefPubMedPubMedCentralGoogle Scholar
  8. Burgoyne RD, O’Callaghan DW, Hasdemir B, Haynes LP, Tepikin AV (2004) Neuronal Ca2+-sensor proteins: multitalented regulators of neuronal function. Trends Neurosci 27:203–209CrossRefPubMedGoogle Scholar
  9. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110:119–122CrossRefPubMedGoogle Scholar
  10. Davis RH, de Serres FJ (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods in Enzymol 17:79–143CrossRefGoogle Scholar
  11. De Castro E, Nef S, Fiumelli H, Lenz SE, Kawamura S, Nef P (1995) Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors. Biochem Biophys Res Commun 216:133–140CrossRefPubMedGoogle Scholar
  12. Deka R, Kumar R, Tamuli R (2011) Neurospora crassa homologue of neuronal calcium sensor-1 has a role in growth, calcium stress tolerance, and ultraviolet survival. Genetica 139:885–894CrossRefPubMedGoogle Scholar
  13. Freitag M, Hickey PC, Raju NB, Selker EU, Read ND (2004) GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol 41:897–910CrossRefPubMedGoogle Scholar
  14. Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J 405:199–221CrossRefPubMedGoogle Scholar
  15. Hamasaki-Katagiri N, Ames JB (2010) Neuronal calcium sensor-1 (Ncs1p) is up-regulated by calcineurin to promote Ca2+ tolerance in fission yeast. J Biol Chem 285:4405–4414CrossRefPubMedGoogle Scholar
  16. Hendricks KB, Wang BQ, Schnieders EA, Thorner J (1999) Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase. Nat Cell Biol 1:234–241CrossRefPubMedGoogle Scholar
  17. Huttner IG, Strahl T, Osawa M, King DS, Ames JB, Thorner J (2003) Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1. J Biol Chem 278:4862–4874CrossRefPubMedGoogle Scholar
  18. Ikura M (1996) Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci 21:14–17CrossRefPubMedGoogle Scholar
  19. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28CrossRefPubMedGoogle Scholar
  20. Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H (2006) Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci USA 103:14871–14876CrossRefPubMedPubMedCentralGoogle Scholar
  21. Ishitani M, Liu J, Halfter U, Kim CS, Shi W, Zhu JK (2000) SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell 12:1667–1677CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jeromin A, Muralidhar D, Parameswaran MN, Roder J, Fairwell T, Scarlata S, Dowal L, Mustafi SM, Chary KV, Sharma Y (2004) N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1. J Biol Chem 279:27158–27167CrossRefPubMedGoogle Scholar
  23. Koh PO, Undie AS, Kabbani N, Levenson R, Goldman-Rakic PS, Lidow MS (2003) Up-regulation of neuronal calcium sensor-1 (NCS-1) in the prefrontal cortex of schizophrenic and bipolar patients. Proc Natl Acad Sci 100:313–317CrossRefPubMedGoogle Scholar
  24. Larrondo LF, Colot HV, Baker CL, Loros JJ, Dunlap JC (2009) Fungal functional genomics: tunable knockout-knock-in expression and tagging strategies. Eukaryot Cell 8:800–804CrossRefPubMedPubMedCentralGoogle Scholar
  25. McCluskey K (2003) The fungal genetics stock center: from molds to molecules. Adv Appl Microbiol 52:245–262CrossRefPubMedGoogle Scholar
  26. McFerran BW, Weiss JL, Burgoyne RD (1999) Neuronal Ca2+ Sensor 1 characterization of the myristoylated protein, its cellular effects in permeabilized adrenal chromaffin cells, Ca2+-independent membrane association, and interaction with binding proteins, suggesting a role in rapid Ca2+ signal transduction. J Biol Chem 274:30258–30265CrossRefPubMedGoogle Scholar
  27. McIlhinney RA (1998) Membrane targeting via protein N-myristoylation. Methods Mol Biol 88:211–225PubMedGoogle Scholar
  28. Moncrief ND, Kretsinger RH, Goodman M (1990) Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J Mol Evol 30:522–562CrossRefPubMedGoogle Scholar
  29. Multani PK, Clarke TK, Narasimhan S, Ambrose-Lanci L, Kampman KM, Pettinati HM, Oslin DW, O'Brien CP, Berrettini WH, Lohoff FW (2012) Neuronal calcium sensor-1 and cocaine addiction: a genetic association study in African-Americans and European Americans. Neurosci Lett 531:46–51CrossRefPubMedPubMedCentralGoogle Scholar
  30. Muralidhar D, Jobby MK, Jeromin A, Roder J, Thomas F, Sharma Y (2004) Calcium and chlorpromazine binding to the EF-hand peptides of neuronal calcium sensor-1. Peptides 25:909–917CrossRefPubMedGoogle Scholar
  31. Nakamura TY, Jeromin A, Smith G, Kurushima H, Koga H, Nakabeppu Y, Wakabayashi S, Nabekura J (2006) Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons. J Cell Biol 172:1081–1091. doi: 10.1083/jcb.200508156 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Nef P (1996) Neuron-specific calcium sensors: the NCS subfamily. In: Celio MR (ed) Guidebook to the calcium-binding proteins. Sambrook and Tooze Publication, Oxford, pp 94–98Google Scholar
  33. Nicholas KB, Nicholas HB, Deerfield DW (1997) GeneDoc: Analysis and visualization of genetic variation. EMBnet News 4:1–4Google Scholar
  34. Olsen HB, Kaarsholm NC (2000) Structural effects of protein lipidation as revealed by LysB29-myristoyl, des (B30) insulin. Biochemistry 39:11893–11900CrossRefPubMedGoogle Scholar
  35. Pall ML, Brunelli JP (1993) A series of six compact fungal transformation vectors containing polylinkers with multiple unique restriction sites. Fungal Genet Newslett 40:59–62Google Scholar
  36. Palma-Guerrero J, Hall CR, Kowbel D, Welch J, Taylor JW, Brem RB, Glass NL (2013) Genome wide association identifies novel loci involved in fungal communication. PLoS Genet 9:e1003669CrossRefPubMedPubMedCentralGoogle Scholar
  37. Permyakov SE, Cherskaya AM, Senin II, Zargarov AA, Shulga-Morskoy SV, Alekseev AM, Zinchenko DV, Lipkin VM, Philippov PP et al (2000) Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites. Protein Eng 13:783–790CrossRefPubMedGoogle Scholar
  38. Piton A, Michaud JL, Peng H, Aradhya S, Gauthier J, Mottron L, Champagne N, Lafrenière RG, Hamdan FF et al (2008) Mutations in the calcium-related gene IL1RAPL1 are associated with autism. Hum Mol Genet 17:3965–3974CrossRefPubMedGoogle Scholar
  39. Podell S, Gribskov M (2004) Predicting N-terminal myristoylation sites in plant proteins. BMC Genomics 5:1CrossRefGoogle Scholar
  40. Pongs O, Lindemeier J, Zhu XR, Theil T, Engelkamp D, Krah-Jentgens I, Lambrecht HG, Koch KW, Schwemer J, Rivosecchi R et al (1993) Frequenin—a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system. Neuron 11:15–28CrossRefPubMedGoogle Scholar
  41. Resh MD (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta (BBA) Mol Cell Res 1451:1–16CrossRefGoogle Scholar
  42. Ryan FJ (1950) Selected methods of Neurospora genetics. Methods Med Res 3:51–75Google Scholar
  43. Ryan FJ, Beadle GW, Tatum EL (1943) The tube method of measuring the growth rate of Neurospora. Am J Bot 30:784–799CrossRefGoogle Scholar
  44. Senin II, Fischer T, Komolov KE, Zinchenko DV, Philippov PP, Koch KW (2002) Ca2+-myristoyl switch in the neuronal calcium sensor recoverin requires differentfunctions of Ca2+-binding sites. J Biol Chem 277:50365–50372CrossRefPubMedGoogle Scholar
  45. Strahl T, Huttner IG, Lusin JD, Osawa M, King D, Thorner J, Ames JB (2007) Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1). J Biol Chem 282:30949–30959CrossRefPubMedGoogle Scholar
  46. Strynadka NCJ, James MN (1989) Crystal structures of the helix-loop-helix calcium-binding proteins. Annu Rev Biochem 58:951–999CrossRefPubMedGoogle Scholar
  47. Tamuli R, Ravindran C, Kasbekar DP (2006) Translesion DNA polymerases Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa. J Biosci 31:557–564CrossRefPubMedGoogle Scholar
  48. Tamuli R, Kumar R, Deka R (2011) Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi. J Basic Microbiol 51:120–128CrossRefPubMedGoogle Scholar
  49. Tamuli R, Deka R, Borkovich KA (2016) Calcineurin subunits A and B interact to regulate growth and asexual and sexual development in Neurospora crassa. PLoS One 11:e0151867CrossRefPubMedPubMedCentralGoogle Scholar
  50. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  51. Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implications. Am Nat 98:435–446CrossRefGoogle Scholar
  52. Weiss JL, Archer DA, Burgoyne RD (2000) Neuronal Ca2+ sensor-1/frequenin functions in an autocrine pathway regulating Ca2+ channels in bovine adrenal chromaffincells. J Biol Chem 275:40082–40087CrossRefPubMedGoogle Scholar
  53. Westergaard M, Mitchell HK (1947) Neurospora V. A synthetic medium favoring sexual reproduction. Am J Bot 34:573–577 CrossRefGoogle Scholar
  54. Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Department of Biosciences and BioengineeringIndian Institute of Technology GuwahatiGuwahatiIndia

Personalised recommendations