Genomics and Applied Biology 2015, Vol. 6, No. 7, 1-8
2
1994). CaM stimulates plasma membrane Ca
2+
-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). Besides interacting
with (Ca
2+
+Mg
2+
) -activated ATPase in isolated
cardiac sarcoplasmic reticulum and RBC membrane,
CaM also interacts with ciliary dynein ATPase of
Tetrahymena
(Blum et al., 1980; Kirchberger and
Antonetz, 1982; Lopes et al., 1990). In plants,
Ca
2+
-activated CaM regulates different Ca
2+
-ATPases
(Peerseen et al., 1997; Harper et al., 1998; Hong et al.,
1999; Chung et al., 2000; Malmström et al., 1997,
2000). 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; Tamuli et al., 2013).
N. crassa
ATPases are found distributed in different branches
during a phylogenetic analysis, NCA1 in ERCA,
NCA2 and NCA3 in PMCA, PMR1 in PMR1, and
PH-7 in ENA branch (Haro et al., 1991; Benito et al.,
2000). Lack of NCA-2 results in slow growth, Ca
2+
sensitivity, female sterility, and accumulation of more
Ca
2+
than the wild-type; indicating that it functions in
the plasma membrane to pump Ca
2+
out of the cell
(Bowman et al., 2011). NCA-2 is more similar to the
PMC-type proteins of animal cell than the Pmc1p in
S.
cerevisiae
that resides in the vacuole (Bowman et al.,
2011). In addition, one of the cation-ATPases
trm-9
,
which is encoded by the gene NCU04898, shows
sequence homology to
spf1
gene of
S. cerevisiae.
SPF1 family ATPases genes are conserved from yeast
to human; however, the functions of these ATPases
remain 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).
To investigate the cellular role of CaM in
N. crassa
,
we used CaM antagonists, TFP and CPZ. Moreover,
we studied two other genes
trm-9
and
nca-2
using
their knockout mutants. We found that the
cmd, trm-9,
and
nca-2
genes
play a role in growth, Ca
2+
sensitivity,
and in acquisition of thermotolerance induced by
heat shock temperature in
N. crassa
.
Materials and Methods
Sequence analysis
BLAST (Altschul et al., 1990) analysis was performed
using software tools available from NCBI
and the proteins
were selected based on
E
value, % identities and
gapes as described previously (Tamuli et al., 2011).
The Conserved Domain Database (CCD) (Marchler-
Bauer et al., 2009) database was used to identify
conserved domains in the protein. The homologue
protein sequences were aligned with ClustalX 1.83
(Thompson et al., 1997) and visualized using GeneDoc
(Nicholas et al., 1997). Phylogenetic trees were
constructed from the aligned sequences using the
minimum-evolution method (Rzhetsky and Nei, 1992),
500 bootstrap replications as test of phylogeny
(Felsenstein 1985) and the software MEGA5 (Tamura
et al., 2011). Promoter region was analyzed by
selecting ~2 kb sequences from upstream of Transcription
Start Site and transferred to MatInspector in
Genomatix software (
de/cgi-
bin//matinspector_prof/mat_fam) to predict transcription
factor binding sites (Quandt et al., 1995).
Strains, growth, crosses maintenance
N. crassa
wild-type strains 74-OR23-1
mat
A
(FGSC
987), 74-OR8-1
mat
a
(FGSC 988), Ca
2+
signaling
mutant strain ∆NCU04898.2::
hph mat
A
(FGSC 1304
0), and ∆NCU04736.2::
hph
mat
A (FGSC 13071)
were generated by the
Neurospora
genome project and
obtained from the Fungal Genetics Stock Center
(FGSC; University of Missouri, Kansas city, MO
64110) (Colot et al., 2006; McCluskey 2010). The
∆NCU04898.2::
hph
∆NCU04736.2::
hph
double mutant
was generated by crossing the individual single
mutant strain, and presence of the knockout alleles
