Growth, Differentiation and Sexuality

66 J.H. Sietsma and J.G.H. Wessels
wouldpermitlargerproteinstodiffusereadilyfrom
this region into the medium. Within this context,
differences in the gradients by which wall components
are extruded into the apical wall could also
account for some of the layering seen in the walls of
fungi, without inferring temporal differences in the
synthesis of these wall components (Wessels 1990).
A similar mechanism of passage of proteins
through the wall could apply for yeasts during
bud growth, particularly during the first polarised
growth phase. Even during the phase of more general
wall expansion, proteins should be pushed into
the wall by the accretion of wall material at the inside.
A coupling between wall expansion and excretion
has been documented in yeasts (Schekman
1985). However, protein translocation over the wall
by the proposed mechanism is expected to be less
efficient than in mycelial fungi where the most apically
excreted proteins would flow continuously to
the outside regions of the wall. In addition, proteins
in yeasts would not be released into the medium if
they became cross-linked to the polysaccharides
of the wall, as seems to be the case for the highmannan
protein fraction of the yeast cell wall (see
The Mycota, Vol. I, 1st edn., Chap. 6, and Vol.
XIII, Chap. 9) and the sexual adhesion protein αagglutinin
(Schreuder et al. 1993). Moreover, the
presence of these structural mannoproteins seems
to impede the release of other proteins into the
medium (Zlotnik et al. 1984; de Nobel and Barnett
1991).
What then is the evidence for protein excretion
occurring mainly at the growing apex? Direct
evidence was obtained by growing colonies of A.
niger and Phanerochaete chrysosporium in a very
thin layer between two porous polycarbonate
membranes (Wösten et al. 1991; Moukha et al.
1993a,b). By this method it was possible to
localise, by autoradiography, both apical wall
growth and the excretion of proteins. Apical
growth was monitored by the incorporation of
radioactive N-acetyl-glucosamine into chitin,
protein synthesis and excretion by the incorporation
of radioactive sulphate and catching
excreted proteins on a protein-binding membrane
underneath the sandwiched colony. In young,
actively growing colonies, chitin labelling occurred
most intensely at apices at the periphery
of the colony, as expected. However, whereas
cytoplasmic proteins were labelled throughout
the colony, protein excretion occurred almost
exclusively in the peripheral growth zone. In A.
niger, the excretion of glucoamylase could be
immunologically detected in this zone, and at the
hyphallevelitcouldbeshownthataconsiderable
portion is actually excreted at hyphal apices.
Another portion of the glucoamylase, however,
is retained for some time in the hyphal wall
and appears to diffuse into the medium only
slowly (Wösten et al. 1991). In A. niger, apical
secretion and transient retaining of protein in
the subapical wall was also demonstrated for
a glucoamylase:green fluorescent protein fusion
(Gordon et al. 2000). Importantly, apical excretion
was also suggested for idiophase enzymes which
are excreted only after growth of the mycelium as
a whole has ceased. In Ph. chrysosporum, Moukha
et al. (1993a,b) found that after cessation of radial
growth of the colony a new growth zone arises in
the centre of the colony, characterized by branching
of the resident hyphae and accompanied by
the formation of mRNAs for lignin peroxidase and
Mn 2+ -dependent peroxidase and the excretion of
these enzymes into the medium, apparently by the
newly formed branches. This reflects the ability of
colonies of these mycelial fungi to redirect growth
even in the absence of external nutrients, by
redistribution of previously assimilated nutrients
(see The Mycota, Vol I, 1st edn., Chap. 9).
The results of these experiments clearly show
that in mycelial fungi there is a tight coupling between
apical wall growth and excretion of proteins
into the medium, both during primary growth and
during idiophase when net growth has come to
a halt. The wall over the growing apex is apparently
the major site for the passage of proteins. If the very
polarised excretion of wall polymers is instrumental
in the passage of proteins over the wall, then this
may explain the tremendous capacity of mycelial
fungi, in contrast to yeasts, to export sometimes
about half of the proteins they make into the external
milieu (Wessels 1993).
VIII. Conclusion
The cylindrical hyphal form is generated by cell
wall synthesis at the apex. The cell wall components,
chitin, (1-3)-β-glucan and (1-3)-α-glucan,
are synthesised separately at the apex, and crosslinked
and modified in subapical regions. In this
way,acellwallisgeneratedwhichisplasticatthe
very apex, expandable by turgor pressure and rigid
at subapical parts of the hypha, resistant to turgor
pressure. At the apex a gradient of wall synthe-