The sequence analysis revealed that NCU04379 gene encodes a Ca2+ and/or CaM binding protein of 190 amino acid residues that shows sequence similarity to the homologues of Neuronal Calcium Sensor-1 (NCS-1) from M. grisea, A. fumigatus, S. japonicus, S. pombe, D. rerio, H. sapiens, M. musculus, X. Laevis, and S. cerevisiae (92, 91, 79, 78, 66, 66, 66, 65, and 59% identity; 95, 94, 91, 90, 82, 82, 82, 82, and 79% similarity; e-values 6e-121, 1e-121, 7e-108, 8e-106, 2e-87, 2e-68, 3e-93, and 4e-64, respectively). Therefore, the NCU04379 encodes a N. crassa homologue of NCS-1. The functions of NCS-1 orthologues were previously identified in S. cerevisiae, M. grisea, A. fumigatus, S. pombe, D. melanogaster, and H. sapiens. In S. cerevisiae, Frq1, the NCS-1 homologue, was found essential for cell growth and viability (Hendricks et al. 1999). The spores of frq1 null mutants were inviable and growth was stopped after three or four divisions. The Frq1 protein, upon Ca2+ binding, changes its conformation and exposes its N-myristoyl group through which the protein physically associates with the N-terminus of phosphatidylinositol-4-OH kinase (Pik1) and contribute to optimal function of Pik1 by assisting with its membrane attachment (Huttner et al. 2003). In M. grisea, null-mutants for ncs-1 like gene, Mg-ncs-1, showed normal growth and pathogenicity similar to their parental strains, but high concentrations of Ca2+ and acidic conditions suppressed their growth (Saitoh et al. 2003). In S. pombe, ncs1 mutant showed nutrition-insensitive sexual development and Ca2+ sensitivity at high concentrations of Ca2+
(Hamasaki-Katagiri et al. 2004). The starvation independent sexual development in ncs1 of S. pombe was suppressed by exogenous cAMP suggesting the involvement of Ncs1p in the adenylate cyclase pathway switched on by a glucose-sensing G-coupled receptor Git3p (Welton and Hoffman 2000). Thus, in contrast to the lethal null phenotype of the budding
yeast homolog Frq1, the ncs1 gene in fission yeast was found non-essential for vegetative growth. In A. fumigatus, ∆ncsA mutant was more resistant to CaCl2 and sensitive to EGTA suggesting the involvement of NcsA, the NCS-1 homologue, in Ca2+ metabolism. Moreover, NcsA was found to support the expression of ion pumps pmcA and pmcB regulating Ca2+
metabolism in the cytoplasm. NcsA was also found to be involved in sterol distribution and polar establishment in the tip of A. fumigatus (Mota Ju´nior et al. 2008). In Drosophila, Frequenin modulates Ca2+ entry through a functional interaction with voltage-gated Ca2+- channel subunit. This regulates neurotransmission release and nerve terminal outgrowth (Dason et al. 2009). In mammals, NCS-1 is involved in anti-apoptotic mechanism in adult motor neurons.It was found to be a novel survival-promoting factor in bilateral dorsal motor nucleus of vagus neurons in adult rats. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressorswhereas the dominant- negative mutant (E120Q) accelerated the cell death (Nakamura et al. 2006). In Danio rerio, ncs-1a gene encoding NCS-1 is required for semicircular canal formation. The knockdown of ncs-1a blocks normal development of non-sensory components of semicircular canals (Petko et al. 2009). In Caenorhabditis elegans, proper calcium signaling via NCS-1 is essential for associative learning and memory (Gomez et al. 2001).
The knockout mutant of N. crassa homologue of NCS-1 displayed slow growth rate and hypersensitivity to Ca2+ stress (Figures 3.2, 3.4, discussed in Chapter 3) which was in contrast to the phenotype of A. fumigatus ∆ncsA mutant that showed resistance to Ca2+ stress.
This result suggested that homologues of NCS-1 in N. crassa and A. fumigatus functions differently with regard to Ca2+ metabolism. Additionally, UV-sensitivity of the
∆NCU04379.2 mutant strain (Figure 3.8, discussed in Chapter 3) uncovered a novel function of N. crassa homologue of NCS-1. This suggested the involvement of N. crassa homologue of NCS-1 in UV-induced DNA damage repair process. UV light absorption may cause DNA damage primarily through formation of cyclobutane pyrimidine dimers (CPD; Lippke et al.
1981) and pyrimidine (6–4) pyrimidone photoproducts (6–4PP; Mitchell and Nairn 1989) leading to induction of DNA repair mechanisms or apoptosis (Lo et al. 2005). It was also reported that overexpression of CaM activates H2AX mediated DNA repair after irradiation and the CaM protein antagonists fully or partially block double-stranded DNA repair in human cells (Wang et al. 2000; Herman et al. 2002; Smallwood et al. 2009).
The site-directed mutational analysis revealed that N-myristoylation in N. crassa homologue of NCS-1 is not essential for its growth and Ca2+ stress tolerance since ncs-1G2A mutant complemented the slow growth and Ca2+ sensitivity phenotypes of the ∆ncs-1 mutant (Figures 4.17, 4.18). However, the ncs-1G2A mutant showed an increased sensitivity to UV like the ∆ncs-1 mutant (Figure 4.19). This result suggested that the N-myristoylation mediated membrane targeting is essential for the NCS-1 role in UV induced DNA damage repair in N. crassa. The R175A mutation in the hydrophobic pocket of N. crassa homologue of NCS-1 complemented growth defect and Ca2+ sensitivity phenotypes of the ∆ncs-1 mutant (Figure 4.20, 4.21). However, the ncs-1R175A mutant was more sensitive to UV than the ∆ncs- 1 mutant (Figure 4.22). This could indicate the molecular basis of the novelty of the NCS-1 protein for its involvement in UV-induced DNA damage and repair process in N. crassa. The E120Q mutation in the EF3 domain was found to be very critical for NCS-1 functions in N.
crassa since ncs-1E120Q mutant did not complement the slow growth, Ca2+ and UV sensitivity phenotypes of the ∆ncs-1 mutant (Figures 4.23, 4.24, 4.25). In addition, ncs-1E120Q mutant displayed slow growth rate that was much lower than the ∆ncs-1 mutant indicating the dominant negative effect of ncs-1E120Q allele in growth.
Thus, this Chapter describes sequence analysis that identified NCU04379 gene as a homologue of NCS-1. In addition, site-directed mutational analysis had identified three critical residues of NCS-1 in N. crassa. A part of this Chapter was published in Journal of Basic Microbiology (Tamuli et al. 2011) and in Genetica (Deka et al. 2011). Genetic interaction of ncs-1 will be discussed in the next Chapter.