Introduction

7. Genomatix software: This is used for promoter analysis of Genomic DNA sequence

3.1 Introduction

The genome analysis of filamentous fungus N. crassa has identified ~10,082 protein coding genes including 48 Ca²⁺-signaling genes that encode for Ca²⁺-channel proteins, Ca²⁺/cation-ATPases, Ca²⁺/H⁺ exchangers, Ca²⁺/Na⁺ exchangers, phospholipase C-δ subtype, CaM and Ca²⁺ and/or CaM binding proteins (Galagan et al. 2003; Borkovich et al. 2004). All Ca²⁺-signaling knockout mutants were generated using a high-throughput gene knockout procedure developed by the Neurospora genome project

(http://www.dartmouth.edu/~neurosporagenome/proj_overview.html; Colot et al. 2006) and obtained from the FGSC (Figure 3.1).

Figure 3.1 Strategy to generate deletion constructs for generating the N. crassa knockout mutants. The 5’ and 3’-flanks of the target open reading frame (ORF) were amplified separately from wild-type with primers 5f with 5r and 3f with 3r. Primers 5r and 3f contained MmeI site (M) and 5’ tails homologous to the hph cassette. In addition, tails of the primers 5f and 3r were homologous to the pRS426 vector. The hph cassette was separately PCR amplified from the pCSN44 vector (Staben et al. 1989) using specific primer pair. The two flanks, the hph cassette, and gapped yeast shuttle vector (pRS426) were co-transformed into the yeast strain FY834 (Winston et al. 1995) to generate the circular construct through homologous recombination. The pooled yeast DNA was used as template with primers 5f and 3r to PCR amplify final linear deletion cassette. The selectable marker hph is transcribed in the antisense direction relative to the direction of transcription of the target gene (Adapted from Colot et al. 2006).

The only CaM in N. crassa is encoded by the NCU04120 gene and CaM appears to be essential for viability (Galagan et al. 2003; Borkovich et al. 2004). Therefore, I studied the cellular role of CaM in N. crassa using two CaM antagonists TFP and CPZ. TFP (https://pubchem.ncbi.nlm.nih.gov/compound/trifluoperazine#section=Top) and CPZ (https://pubchem.ncbi.nlm.nih.gov/compound/chlorpromazine) are typical antipsychotic of the phenothiazine chemical class (Figure 3.2).

Figure 3.2: Structure of the CaM antagonists. (A) TFP and (B) CPZ (Adapted from PubChem).

The CaM antagonists compete with Ca²⁺ for binding to the CaM that consequently inhibit Ca²⁺/CaM signaling (Ahn and Suh 2007a). In its activated (Ca²⁺-bound) conformation, CaM exposes hydrophobic residues to the solvent, enabling binding to a target, either a protein or an inhibitor. The binding process itself requires a large conformational change involving the unfolding of the central helix in order to allow for rotation of the C- terminal domain to form the binding site. The binding of CaM antagonists, particularly the phenothiazine to the lipophilic domain of CaM is well documented, to elucidate the role of CaM in cellular regulation (Roufogalis 1985). The crystal structure of CaM bound to TFP has been determined and refined to a resolution of 2.45 Å(Cook et al.

1994), but the crystal structure of Ca²⁺-CaM-TFP complex has been determined to 2 Å resolution in 1994 (Vandonselaar et al. 1994). Only one TFP is bound to CaM and that is sufficient to cause distortion of the central α-helix and juxtaposition of the N- and C- terminal domains as seen in CaM-polypeptide complexes (Cook et al. 1994).

Inactivation of Ca²⁺-CaM by TFP is possibly due to this major tertiary structural

alteration in Ca²⁺-CaM that is initiated and stabilized by drug binding. The drug makes only a few contacts with one residue in the N-terminal domain and extensive contacts with residues in the C-terminal domain of CaM. Two hydrophobic binding pockets, created by amino acid residues adjacent to Ca²⁺-coordinating residues, form the key recognition sites on Ca²⁺-CaM for both inhibitors and target enzymes (Vandonselaar et al. 1994). In addition, CPZ is a phenothiazine with actions similar to TFP. The spin- labelled derivative of chlorpromazine (CPZSL) was used to probe the phenothiazine binding site of CaM by electron spin resonance (ESR) spectrometry. The completion of the spectroscopic changes requires the presence of 4 Ca²⁺ ions per CaM molecule.

CPZSL has a high sensitivity to various effectors in the environment of the drug binding site (Rainteau et al. 1984). Both CaM activated muscle MLCK and pea NAD⁺ kinase act in a Ca²⁺-dependent manner and these activities were inhibited by TFP or CPZ (Nakamura et al. 1984). TFP and CPZ were shown to affect different cell functions in N. crassa, such as shortening of period length of the conidiation rhythm, light induced phase shifting and activation of chitin synthase enzyme in N. crassa (Suzuki et al. 1996;

Sadakane and Nakashima 1996; Suresh and Subramanyam 1997).

CaM interact with various proteins and enzymes to amplify the Ca²⁺-signaling. One of the targets of CaM is the Ca²⁺ -ATPase, a Ca²⁺ pump that help in fine tuning of Ca²⁺ homeostasis in cells by pumping Ca²⁺ out of cells. CaM stimulates plasma membrane Ca²⁺-ATPase (PMCA) activity by binding to an autoinhibitory domain of PMCA. The CaM-binding domain is located near the C-terminus of PMCA (Osborn et al. 2004; Giacomello et al. 2013). In N. crassa, nine ATPases have been identified and they possess conserved cation transporter/ATPase domain in the proteins (Galagan et al.

2003; Borkovich et al. 2004). Lack of the nca-2 (NCU04736) gene, a Ca²⁺-ATPase, results in slow growth, female sterility and accumulates more Ca²⁺ than the wild-type, indicating that it functions in the plasma membrane to pump Ca²⁺ out of the cell (Bowman et al. 2011). In addition, one of the cation-ATPase, trm-9 (NCU04898) shows sequence homology to SPF1 ATPase of S. cerevisiae. SPF1 family ATPases genes conserved from yeast to human, however, the function of these ATPases is unclear. SPF1 is not essential for cell viability and its substrate specificity is unknown and loss of SPF1 may perturb homeostasis of ions that affects modification and sorting of proteins in the secretory pathway of yeast (Cronin et al. 2000; Suzuki 2001). However, mechanism of trm-9 functions remains unknown in N. crassa. In order to understand trm-9 functions

and its relation with other ATPases including the one encoded by the nca-2 gene, I have studied their genetic interactions.

Thus, in this Chapter, I describe the cellular role of CaM based on the effect of TFP and CPZ and functions of nca-2 and trm-9 in N. crassa.