Growth, Differentiation and Sexuality

shock,lowosmolarityaswellasotherfactorsare
believed to be key environmental signals which activate
this pathway. Several cell membrane proteins
(Mid2p, Mtl1p, Wsc1-4p, Zeo1p) have been suggested
to function as sensors for the activation
of the cell integrity pathway in S. cerevisiae (Zu
et al. 2001; Green et al. 2003). The regulation of the
PKC1 signal pathway is not unexpectedly complex,
and may include several other regulatory proteins.
For example, in S. cerevisiae, the Pkh1/2p homologues
of the mammalian 3-phosphoinositide protein
kinase can regulate both the PKC1 and YPK1
signal pathways (Zhang et al. 2004). The regulation
of these pathways requires sphingolipid long
chain bases which act via two specific regulators,
Pil1p and Lsp1p. There is also recent data from
S. cerevisiae that, of these many signals activating
the PKC1 pathway, not all are integrated as a “topdown”
mechanism which depends upon GTP loading
of Rho1p (Harrison et al. 2004). These authors
show that strains lacking Pkc1p and Bck1p are still
able to activate Slt2p when cells were stressed by
heat shock, but activation of Slt2p occurs differently
under conditions of actin depolymerisation
or hyposmotic shock. The authors conclude that
stress conditions provide lateral inputs into this
regulatory pathway, rather than in a “top-down”
linear manner. One example of this “lateral” interaction
is Knr4p, whose interaction with Slt2p is essential
for signalling through the cell wall integrity
pathway (Martin-Yken et al. 2003).
The MKC1 gene (MAP kinase from C. albicans)
isahomologueoftheS. cerevisiae SLT2. Although
it is not essential for the growth of the organism,
several phenotypes were observed which
indicated a sensitivity of the deletion mutant to
growth at 42 ◦ C or heat shock at 55 ◦ C, a sensitivity
of cells to caffeine which could be reversed
by the addition of 1M sorbitol and to β1,3 glucan
inhibitors, and a lower susceptibility to complex
lytic enzymes such as the glusulase enzyme and
nikkomycin (Navarro-Garcia et al. 1995, 1998). Differences
between the mutant and wild-type cells in
the kinetics of cell wall precursor incorporation
were not observed, and a comprehensive analysisofcellwallcompositiononlyrevealedminimal
changes in total chitin. Interestingly, a mannoprotein
epitope (1B12) was more highly expressed in
the mutant but further analysis of this phenotype
was not pursued (Navarro-Garcia et al. 1995, 1998).
These phenotypes indicate that the role for MKC1
in cell wall biosynthesis is less in C. albicans than
in S. cerevisiae.InMagnaporthe grisea,thehyphae
Fungal Cell Wall 93
of the mps1/slt2 mutant are hypersensitive to cell
wall-degrading enzymes, thinner and uneven, indicating
that wall changes must accompany the gene
deletion (Xu and Hammer 1996; Dixon et al. 1999).
The deletion of MPK1 in C. neoformans induced
adefectingrowthat37 ◦ C which was overcome
by the addition of sorbitol. Along with this observation,
the mpk1 mutant exhibited enhanced sensitivity
to nikkomycin, and partial sensitivity to
caspofungin which was further enhanced in a double
mutant of mkc1/cnb1, the latter encoding the
B subunit of the calcineurin protein. In wild-type
cells, Mpk1 was phosphorylated when cells were
stressed with Calcofluor white. The growth phenotype,
and the sensitivity of the mutants to cell wall
perturbing drugs again point to the major role of
the cell integrity pathway with regard to cell wall
synthesis.
3. TOR and Calcineurin Signalling Pathway
The TOR (target of rapamycin) kinases are highly
conserved proteins found in organisms from
yeasts to humans (Crespo and Hall 2002). They
are originally characterized and named according
to the observation that the immunosuppressive
drugs rapamycin and tacrolimus (FK506) bind to
a protein called immunophilin (Fkbp12p, FK506binding
protein of 12 kDa), which in turn binds
and inhibits the kinase TOR. In S. cerevisiae,there
are two TOR-encoding genes, TOR1 and TOR2
(Crespo and Hall 2002). Both share a common
function of activation of translation initiation and
cell cycle progression in response to nutrients. In
addition, TOR2 mediates the cell cycle-dependent
actin polarization to the bud site of dividing cells.
This event is initiated through Rom2p activation
of Rho1p and subsequent signalling via the
PKC-MAP kinase pathway. Thus, Tor2p function
cross-talks with the cell integrity pathway (Crespo
and Hall 2002). The TOR kinases are members
of the phosphatidylinositol 3-kinase (PI-3K)
superfamily which are critical for the regulation of
cell growth and differentiation.
Calcineurin is a Ca 2+ -calmodulin-activated
serine/threonine phosphatase (Lengeler et al. 2000;
Rohde et al. 2001) which consists of a catalytic
AsubunitandaCa 2+ -binding regulatory subunit.
This pathway connects Ca 2+ -dependent signalling
with many cellular responses, including TOR
functions (Fig. 5.12; Sugiura et al. 2002). In S. cerevisiae,
Cch1/Mid1 are putative calcium-sensitive
sensors upstream of the calcineurin pathway