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