Growth, Differentiation and Sexuality

58 J.H. Sietsma and J.G.H. Wessels
fact, chitin synthase 3, thought to be responsible in
this yeast for most chitin synthesis in vivo, appears
non-zymogenic (Orlean 1987; Shaw et al. 1991;
Bulawa 1992). Another model for the regulation
of chitin synthase activity, based on the study
of secretory mutants in S. cerevisiae, hasbeen
proposed, involving a specialised mechanism of
vesicle sorting (Ziman et al. 1996). In this model,
chitin synthase is maintained inside specialised
vesicles (chitosomes) and shuttled between a store
ofthesevesicles,whereitisinaninactiveform,and
thesiteoffunction,whereitbecomesactivated
by insertion into the cytoplasmic membrane.
Inactivation occurs by endocytosis. The enzyme is
initially not degraded but stored inside chitosomes
until needed, and eventually shuttled to the
vacuole and degraded by proteolysis (Chuang and
Schekman 1996; Valdivia et al. 2002).
This is in agreement with the finding in earlier
studies that isolated protoplasts from different
parts of hyphae start chitin synthesis without delay
when transferred to regenerating conditions, even
in the presence of antibiotics inhibiting protein
synthesis (Sonnenberg et al. 1982). During the formation
of protoplasts, insertion of preformed cytoplasmic
chitin synthase into the plasma membrane
may have occurred. Inhibitors of protein synthesis
have also little influence on the rate of chitin synthesis
in growing hyphae; they only induce a shift
of chitin synthesis from apical to subapical regions
of the hyphae (Katz and Rosenberger 1971a; own
unpublished data), indicating a slow turnover of
chitin synthase molecules.
Being an integral membrane protein, the lipid
environment can be surmised as important for
regulating the activity of chitin synthase. Arrhenius
plots show clear transition points in this activity,
and delipidification inactivates the enzyme
whereas addition of phospholipids restores the activity
of partially delipidified chitin synthase (Duran
and Cabib 1978; Vermeulen and Wessels 1983;
Montgomery and Gooday 1985). Fungicides which
interfere with sterol synthesis (imidazole derivatives)
cause irregular deposition of chitin (Kerkenaar
and Barug 1984), while polyene antibiotics,
known to interact with sterols, inhibit chitin synthesis
by chitosomes in vitro (Rast and Bartnicki-
Garcia 1981). These data agree with the notion that
the insertion of chitin synthase into the lipid environment
of the plasma membrane in itself could
result in activation of the enzyme.
An unexpected mode of chitin synthesis
regulation was recently proposed in yeast (Lagorce
et al. 2002). This mode of regulation is based on
the availability of the substrate for chitin synthase,
N-acetylglucosamine. The expression of the GFA1
gene, which is directly involved in glucosamine
metabolism, is increased when more chitin is
required,inasortofsalvagemechanismwhen
the cell wall is weakened by the inhibition of the
synthesis of wall components other than chitin. In
earlier studies with mutants of A. nidulans in which
cell wall rigidity was affected, chitin content of the
wall was directly correlated to mutations in genes
involved in the synthesis of N-acetylglucosamine
(Katz and Rosenberger 1971b; Borgia and Dodge
1992). It has recently been found that also in
the mycelial fungi Aspergillus niger, Penicillium
chrysogenum and Fusarium oxysporum, expression
of the homologue of GFA1 was increased during
stress conditions caused by cell wall weakening
(R. Damsveld and A. Ram, unpublished data),
indicating that this mechanism of regulating chitin
synthesis could be a more general phenomenon.
2. Chitin Modifications
The product of chitin synthase is a homopolymer
of (1-4)-β-linked N-acetylglucosamine. In vitro,
the polymer chains spontaneously crystallise by
forming inter- and intra-molecular hydrogen
bonds. X-ray diffraction then shows a crystalline
configuration known as α-chitin, in which chains
are presumably antiparallel (Minke and Blackwell
1978). Because newly formed chitin is very sensitive
to modifying enzymes, e.g. chitinase and
chitin deacetylase (Davis and Bartnicki-Garcia
1984; Vermeulen and Wessels 1984, 1986), it was
concluded that there is a time lapse between
enzymic synthesis and crystallisation. On the
other hand, modification of chitin, e.g. by deacetylation
or linkage to other polysaccharides, would
likely interfere with the crystallisation process. In
growing regions, therefore, where chitin synthesis
occurs, competition may exist between crystallisation
and modification. In most fungal species,
the modification of chitin after synthesis probably
accounts for the fact that, in mature hyphal walls,
crystalline chitin can rarely be detected by X-ray
diffraction. Crystallisation of chitin can be prevented
experimentally by addition of compounds
which strongly adhere to the native chains, e.g. the
optical brightener calcofluor white and congo red.
These compounds maintain chitin in a reactive
form which is susceptible to the action of dilute
acids and chitinase (Vermeulen and Wessels 1984;