Growth, Differentiation and Sexuality

their structural role in the cell wall remains controversial.
In yeast, several studies have reported
that numerous proteins originally anchored to the
plasma membrane by a glycosylphosphatidyl inositol
(GPI) anchor subsequently become covalently
linked to β1,3 glucans through a β1,6 glucan linker
(Kapteyn et al. 1995, 1996; Kollar et al. 1997). The
putative localisation of the GPI-anchored proteins
of S. cerevisiae in either the membrane or the cell
wall has been investigated initially by Caro et al.
(1997) and Hamada et al. (1998), and confirmed
recently in Candida glabrata by Frieman and Cormack
(2004). About half of the S. cerevisiae GPI
proteins analysed were thought to be covalently
bound to the cell wall. These data led the group of
Klis to develop a model where the cell wall proteins
wouldhaveanimportantroleinthecross-linkingof
the different cell wall polysaccharides (Smits et al.
1999; Klis et al. 2002). However, the same research
team showed that disruption of genes coding for
the major GPI anchor proteins bound to cell wall
did not affect growth, suggesting that these proteins
were not essential for fungal growth (van der
Vaart et al. 1995). Similarly, disruption of the PIR
genes did not result in mutants with altered growth
(Mrsa and Tanner 1999). Moreover, if the mannoproteins
account for 40%–50% of the cell wall dry
weight (Klis 1994), and if polypeptides have an averageMrof30–60kDa
with 150 mannose unit per
glycoprotein, this means that the proteins should
account for 10%–20% of the cell wall dry weight.
Such an amount of protein has never been found in
the cell wall after SDS treatment, suggesting that the
vast majority of the cell wall-associated proteins is
not covalently bound. In addition, other fungi such
as A. fumigatus do not have proteins covalently
bound to the polysaccharides of the mycelial cell
wall (Bernard et al. 2002). In A. fumigatus, some
of the proteins, such as PhoAp, are tightly bound
to the cell wall in the absence of covalent linkages,
and can be released only as a soluble protein
by a β1,3 glucanase treatment which loosens the
polysaccharide net to which this protein is associated
(Bernard et al. 2002; Latgé et al. 2005). Similarly,someofthePIRgenescodeforproteinswhich
do not have a cell wall localisation to fulfil their
biological function, although they require a mild
alkali treatment to be released from the cell wall
(Vongsamphanh et al. 2001). Strong non-covalent
bonds could indeed occur for several putative cell
wall-bound proteins in yeast, since the identification
of the chemical linkages between the protein
and the polysaccharide (the ultimate proof of such
Fungal Cell Wall 79
covalent linkages) has been assessed only for the
GPI-anchored protein TIP1 and one unknown protein
in S. cerevisiae (Kollar et al. 1997; Fuji et al.
1999). These data show also that the postulated “release
by β1,3 glucanase equals covalent linkage to
the cell wall polysaccharide” is evidently not always
true. This chemical analysis of the cell wall proteins
in A. fumigatus was confirmed by a recent comparative
genomic analysis of the genes coding for
GPI-anchored proteins. Among the GPI proteins
common to S. cerevisiae and A. fumigatus,onlysix
families of GPI proteins in A. fumigatus were orthologues
of yeast GPI proteins: SPS2, GAS/GEL, DFG,
PLB, CRH and YPS (Merkel et al. 1999; Olsen et al.
1999; Mouyna et al. 2000a; Rodriguez-Pena et al.
2000; Bernard et al. 2002; Kitagaki et al. 2002, 2004;
Tougan et al. 2002; Spreghini et al. 2003; Coluccio
et al. 2004; Eisenhaber et al. 2004). Five of
these families were classified as membrane-bound
GPI proteins in yeast (Caro et al. 1997; Hamada
et al. 1998; de Groot et al. 2003), and the covalent
association of the sixth one, CRH, with the cell
wall could be questioned, since this family has sequence
signatures suggesting a β1,3 glucanase activity
(Rodriguez-Pena et al. 2000). This genomic
analysis is in agreement with a proteome study
of membrane GPI-anchored proteins of A. fumigatus
(Bruneau et al. 2001). Four of the families
mentioned above (SPS2, CRH, GEL/GAS and DFG)
havebeenshowntobeassociatedwithcellwallconstruction,
some of them having enzymatic activities
such as β1,3 glucanosyltransferases (Mouyna
et al. 2000a,b; Rodriguez-Pena et al. 2000; Kitagaki
et al. 2002, 2004; Tougan et al. 2002; Spreghini et al.
2003) which do not require any covalent linking to
the cell wall polysaccharides. By contrast, all the
polysaccharide-covalently bound proteins in yeast
such as Flo1p, Fig1p or Aga1p for S. cerevisiae or
Als1p and Epa1p in Candida albicans and Candida
glabrata are involved in cell–cell adhesion, mating,
or adhesion to host cell surfaces (Frieman and
Cormak 2004; Verstrepen et al. 2004). The covalent
binding of proteins to polysaccharides is a way
for the protein to remain at the surface of the cell
wall where it has to bind directly to its ligand to
fulfil a biological function. No genes coding for
PIR proteins are found in the genome of A. fumigatus.
The comparative chemogenomic data mentioned
above suggest that proteins do not have the
role of linkers between cell wall polysaccharides
often proposed for the establishment of the threedimensional
polysaccharide network of the yeast
cell wall (Kapteyn et al. 1999; Smits et al. 1999).