Growth, Differentiation and Sexuality

84 J.P. Latgé and R. Calderone
(Lesage et al. 2004); the third one being a putative
transporter (H. Bussey, personal communication).
Neither the single FKS1 or FKS2 disruptions was
lethal. In contrast to yeasts, all filamentous Ascomycete
moulds sequenced to date have one FKS
orthologue. In A. fumigatus, FKS1 is unique and
essential, as shown in a diploid background after
haploidization following transformation (Firon
et al. 2002) or by RNAi (Mouyna et al. 2004). In S.
pombe,fourgenesBGS1-4 sharing homology with
β1,3-GS catalytic subunits have been identified. Of
these, Cps1p/Bgs1p is an essential gene presumably
involved in the assembly of the septum β1,3
glucan (Ishiguro et al. 1997). Bgs1p is localised to
the cell division site in a manner dependent on
the actomyosin ring and the septation-inducing
network (Liu et al. 1999). In addition, Bgs1p has
been localised to the site of growth (Cortes et al.
2002; Liu et al. 2002). Bgs2p is a sporulationspecific
GS required for proper ascospore wall
maturation (Liu et al. 2000; Martin et al. 2000).
bgs2Δ sporulating diploids show a defect in GS
activity and fail to assemble the cell wall properly,
resulting in a failure to develop viable ascospores.
bgs3 and bgs 4 are essential for cell growth (Martin
et al. 2003; Ribas, unpublished data). Bgs3p cannot
be substituted by overproduction of any of the
other GS homologues, suggesting a unique role for
Bgs3p in the biosynthesis of β1,3 glucans where
new cell wall deposition is necessary. Like Bgs3p,
Bgs4p localises to the growing pole and septum.
Although β1,3-GS isoforms perform a distinct
function,itisalsopossiblethatalltheseisoforms
have overlapping roles in cell wall assembly, as in
S. cerevisiae.
Genome surveys show that the FKS genes are
reasonably well conserved across all fungal genera.
Homologues of FKS1 have been found in all fungi.
Hydropathy analysis of the large protein encoded
by all FKS family members predicts a localisation
within the plasma membrane, with as many as 16
transmembrane helices. A central hydrophilic domain
of about 580 amino acids displays a remarkable
degree of identity (> 80%) among all known
FKS protein sequences (Douglas 2001). It has been
proposed that this region is located on the cytoplasmic
face of the plasma membrane and must have
some essential, conserved function. Two aspartate
residues at positions D392 and D441 in this region
have been recognized as essential for function of
the glucan synthase (Douglas 2001). If members
of the Fks family of proteins provide the catalytic
centre of β1,3 glucan synthase, then they represent
a significant divergence from known glycosyltransferases
which use a nucleotide-diphospho (NDP)sugarasasubstrate.NeitheroftwoproposedUDPglucose
binding sites, (R/K)XGG implicated from
glycogen synthases and D,D,D35QXXRW from hydrophobic
cluster analysis, is found in Fks1p (Douglas
2001). The gaps in our knowledge of the β1,3glucan
synthase mechanism raise many questions.
In the absence of purification to homogeneity of the
enzyme, the first provocative question is whether
Fks1p is the true catalytic subunit of the glucan
synthase or only one of the members of the glucan
synthase complex. Other key questions are similar
to the ones which can be asked for chitin synthesis:
can synthesis be initiated de novo, or does
it require a glucan acceptor? Does polymerisation
proceed from the non-reducing end of the growing
chain? Are glucose residues added as monomers or
as disaccharide units?
b) Regulation
Rho1p-GTPase, which controls β1,3 glucan
synthase, is regulated by switching between
a GDP-bound inactive state and a GTP-bound
activestatewithconformationalchanges(Weietal.
1997; Fig. 5.8). Only mutations in this gene lead
to a growth-deficient phenotype in moulds (Guest
et al. 2004). After synthesis in the ER, Rho1p is
geranylgeranylated, a modification required for
attachment of Rho1p to the membrane and for
transport (Inoue et al. 1999). Geranylgeranylated
Rho1p and Fks1p are transported to the plasma
membrane as an inactive complex through the
classical secretory pathways (Abe et al. 2003).
Rho1p is activated on its arrival at the plasma
membrane by Rom2p, the GDP/GTP exchange
factor of Rho1 which is localised only at the plasma
membrane. This activation, and the movement of
Fks1p on the plasma membrane are required for
proper cell wall β1,3 glucan localisation (Utsugi
et al. 2002). Since Rho1 is a key regulator of fungal
growth, it is not surprising that it is submitted
to multiple regulators. Accordingly, numerous
Rho1p regulators acting downstream on glucan
synthesis have been identified, although their role
is not fully understood. Besides the GTP-exchange
factors (GEF) Rom2p and Rom1p, other GTPase
activating proteins (GAPs) exist, such as Bem2p,
Sac7p and Lrg1p (Heinisch et al. 1999; Watanabe
et al. 2001; Schmidt and Hall 2002; Calonge et al.
2003; Fitch et al. 2004), which down-regulate β1,3
glucan synthesis. A multicopy suppressor screen