European Journal of Cell Biology 90 (2011) 759–769

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European Journal of Cell Biology

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Review

The cellular basis of chitin synthesis in fungi and insects:

Common principles and differences
Hans Merzendorfer ∗
University of Osnabrück, Faculty of Biology and Chemistry, Department of Animal Physiology, Barbarastrasse 11, 49076 Osnabrück, Germany

a r t i c l e i n f o a b s t r a c t

Keywords: Chitin is a polymer of N-acetylglucosamine, which assembles into microfibrils of about 20 sugar chains.
Chitin These microfibrils serve as a structural component of natural biocomposites found in cell walls and spe-
Chitin synthase cialized extracellular matrices such as cuticles and peritrophic membranes. Chitin synthesis is performed
Chitin metabolism
by a wide range of organisms including fungi and insects. The underlying biosynthetic machinery is highly
Cell wall
conserved and involves several enzymes, of which the chitin synthase is the key enzyme. This membrane
Cuticle
Glycosyltransferase integral glycosyltransferase catalyzes the polymerization reaction. Most of what we know about chitin
Peritrophic matrix synthesis derives from studies of fungal and insect systems. In this review, common principles and dif-
ferences will be worked out at the levels of gene organization, enzymatic properties, cellular localization
and regulation.
© 2011 Elsevier GmbH. All rights reserved.

Introduction In its pure form, chitin is a linear homopolymer of N-

After cellulose, chitin is the earth’s second most abundant
organic compound, and is synthesized by a broad variety of organ-
isms of different taxonomic groups. Chitin is found not only in
arthropods including insects, arachnids and crustaceans, but also
in lower invertebrates such as sponges, coelenterates, nematodes
and molluscs (Merzendorfer, 2009). In addition, it is present in
many microbes including fungi, protists and algae. Even some
rhizobial bacteria are known to secrete fatty acid-linked chitin
oligomers that function as nodulation signals triggering symbiotic
responses in leguminous host plants (Peters, 1997). For unknown
reasons, the ability to produce chitin has been lost at the root of
the deuterostome lineage. In many true fungi (eumycota), chitin
replaces cellulose as the structural polysaccharide stabilizing the
cell wall (Latgé, 2007). In invertebrates, chitin usually serves
as a fibrillar component of mechanically resilient biocomposites
such as eggshells, cuticles, cuttlebones, shell and sponge scaffolds
(Merzendorfer, 2009). However, it can support also very mucous
extracellular matrices such as peritrophic membranes functioning
as protective linings in the intestinal tract (Hegedus et al., 2009).
Due to its unique physicochemical properties chitin and its deacety-
lated form (called chitosan) are attracting increasing interest as
raw materials for the chemical and pharmaceutical industries. In
addition, more recent studies revealed that chitin molecules of
appropriate lengths can elicit immune responses (Lee et al., 2008).
∗ Tel.: +49 541 9693502; fax: +49 541 9693503.
E-mail address: merzendorfer@biologie.uni-osnabrueck.de
acetylglucosamine (GlcNAc) being linked by ␤-1,4 glycosidic bonds
(Muzzarelli, 1999). However, as chitin from natural sources usually
contains a varying percentage of deacetylated glucosamine (GlcN)
residues, it is ultimately an heteropolymer of GlcNAc and GlcN
residues (Fukamizo et al., 1986). More strikingly, chitin exists in dif-
ferent crystalline forms known as ␣-, ␤- and ␥-chitin, which have
different physicochemical properties (Kramer and Koga, 1986).
The by far most abundant form is ␣-chitin, which is composed
of microfibrils consisting of about twenty single chitin chains that
are arranged in an antiparallel fashion. This arrangement allows
tight packaging and the formation of numerous intra- and inter-
molecular hydrogen bonds resulting in an immensely high tensile
strength (Kameda et al., 2005; Rudall and Kenchington, 1973). In
the inner fungal cell wall, the microfibrils made of ␣-chitin serve
in compensating the cells’ turgor pressure. In insect cuticles, they
are embedded in a proteinaceous matrix, forming more or less pla-
nar laminae. As layers of chitin and proteins are added, the laminae
are cross-oriented relative to one another at a constant angle and
form helicoidal stacks (Bouligand, 1972). In special cases plywood-
like cuticles can be formed in this way, which are mechanically
extremely robust.
Common to all organisms capable of chitin formation is a
conserved cellular machinery converting sugars into linear chitin
chains, which are then secreted into the extracellular space, assem-
bled into microfibrils, and organized in the extracellular matrix (cell
walls, cuticles, peritrophic matrices). Different cell types are capa-
ble of chitin synthesis. In fungi these cells include vegetative and
sporulating cells (Lesage and Bussey, 2006), and in insects epider-
mal, tracheal and midgut cells (Merzendorfer and Zimoch, 2003). In

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760 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769

Fig. 1. The chitin synthesis pathway in fungi and insects. Glycogen is converted by glycogen phosphorylase (1) to glucose-1-P, which is either fed into glycolysis or used for
trehalose synthesis. Trehalose in turn can be mobilized by hydrolysis to glucose catalyzed by trehalase (2). The conversion of glucose to fructose-6-P involves hexokinase (3)
(EC 2.7.1.1), phosphoglucomutase (4) (EC 5.4.2.2), and glucose-6-P isomerase (5) (EC 5.3.1.9). From fructose-6-P the chitin biosynthetic pathway branches off, with the first
enzyme catalyzing this branch being glutamine-fructose-6-phosphate amidotransferase (6) (GFAT, EC 2.6.1.16). The reaction catalyzed by GFAT converts fructose-6-P into
glucosamine-6-phosphate by transferring the ammonia from the co-substrate l-glutamine and isomerizing the resulting fructosimine-6-phosphate. Next, an acetyl group
from coenzyme A is added by glucosamine-6-P acetyltransferase (7) (EC 2.3.1.4) to obtain N-acetylglucosamine(GlcNAc)-6-P, whose phosphate is then transferred from the
C-6 to the C-1 position catalyzed by phosphoacetylglucosamine mutase (8) (EC 5.4.2.3). The resulting GlcNAc-1-P, finally, is uridinylated by UDP-GlcNAc pyrophosphorylase
(9) (EC 2.7.7.23) yielding UDP-GlcNAc which serves as a substrate for the chitin synthase (10) (CHS, EC 2.4.1.16), a membrane-integral glycosyltransferase transferring the
sugar moiety of UDP-GlcNAc to the growing chitin chain. Chitin is degraded by chitinases (11) (EC 3.2.1.14) and N-acetylglucosaminidases (12) (EC 3.2.1.52) yielding GlcNAc
which can be reused for chitin biosynthesis.

multicellular and many unicellular organisms chitin is secreted in
a polarized fashion. Fungal cells deposit most of the chitin at their
growth sites (sites of emerging bud, bud neck, hyphal tips), while
insect epithelial cells deposit chitin exclusively at their apical sides
facing the external environment (body surface, gut and tracheal
lumen).
Many aspects of chitin synthesis and degradation have been
investigated over the past years raising our understanding of the
underlying cellular machinery. Chitin synthesis is best charac-
terized in fungi and insects. In this review cellular aspects of
chitin synthesis and its regulation will be addressed and compared
between fungi and insects.
A common biosynthetic pathway towards chitin
The general pathway of chitin synthesis is highly conserved from
fungi to insects and involves a defined number of enzymatic reac-
tions that convert different sugars into a polymer of GlcNAc (see
Fig. 1). The sugar source is glucose or its storage compounds glyco-
gen or trehalose (Becker et al., 1996; Francois and Parrou, 2001).
The pathway can be divided into three sets of sub-reactions. The
first set of sub-reactions leads to the formation of the amino sugar
GlcNAc, the second set follows a variant of the Leloir pathway
yielding the activated amino sugar UDP-GlcNAc, and the last sub-
reaction involves the polymerization of chitin using UDP-GlcNAc
as the activated sugar donor. The first two sub-reactions occur in
the cytoplasm and the third one at specialized microdomains of
the plasma membrane. These microdomains include plasma mem-
brane regions at the emerging bud of yeast cells (Elorza et al.,
1983), and at hyphal tips and cross-walls of filamentous fungi
(Watanabe et al., 2005). In insects, the apical plasma membrane
plaques of epidermal cells and the apical microvillar membranes of
midgut cells can be considered as microdomains associated with
chitin synthesis (Locke, 2001; Zimoch and Merzendorfer, 2002).
The rate-limiting enzyme in the first set of sub-reactions appears
to be glutamine-fructose-6-phosphate amidotransferase (GFAT, EC
2.6.1.16). The critical enzyme in the second sub-reaction is UDP-
N-acetylglucosamine pyrophosphorylase (UAP, EC 2.7.7.23), and in
the last sub-reaction the chitin synthase (CHS, EC 2.4.1.16). Not sur-
prisingly, these three enzymes appear to be highly regulated and
determine the rate of chitin synthesis. The reaction catalyzed by
GFAT can be considered as the first committed step of the chitin
biosynthetic pathway, but the last reaction catalyzed by CHS is the
only one, which is specifically associated with chitin biosynthesis.
For this reason CHS is the key enzyme of this pathway, and this is
why it is of special interest in the context of this review.
CHS encoding genes in fungi and insects
Fungal genome sequencing projects revealed between two and
up to more than twenty CHS genes per species, which were cate-
gorized into five (reviewed in Ruiz-Herrera and Ortiz-Castellanos,
2010) or seven classes (reviewed in Lenardon et al., 2010b; Roncero,
2002) (see also Fig. 2); the latter classification will be used in this
review. Further analyses of the deduced amino acid sequences
revealed that CHS enzymes belong to one of two families, which dif-
fer in their activity-related motif. Family I enzymes contain classes
I–III and exhibit the chitin synth 1 motif (PF01644), while family
II enzymes contain classes IV–VII and exhibit the chitin synth 2
motif (PF03142). While class I, II and IV CHSs are found in yeast
and filamentous fungi, class III, V, VI and VII CHSs are found exclu-
sively in filamentous and some dimorphic fungi. Class V and some
class VI and VII enzymes contain N-terminal myosin-like motor
domains (MMDs), which have been implicated in polarized growth
and virulence but not in vesicle motility (Takeshita et al., 2006;
Treitschke et al., 2010). The gene sizes differ significantly between
the different CHS classes particularly in basidiomycetes, where the
relative positions and lengths of existing introns contribute to these
variations (Mehmann et al., 1994). The functional diversity of CHS
gene products is only partially understood. The multiplicity of CHS
genes was initially interpreted in terms of redundant functions in
cell wall synthesis. However, in recent years data have accumu-
lated suggesting functional diversification at least to some extent.
Most studies performed in fungi have focused on chitin synthesis
in the baker’s yeast, Saccharomyces cerevisiae. This yeast’s genome
contains three CHS genes (ScCHS1, ScCHS2, and ScCHS3), which are
expressed in a regulated fashion during the cell cycle. ScChs1, a
class I enzyme, replenishes chitin in the birth/bud scar after cytoki-
nesis and hence acts as a sort of repair function. The class II enzyme
ScChs2 is involved in synthesis of the primary septum and the
 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769
Fig. 2. Phylogenetic tree of CHS enzymes from fungi and insects. The neighbor join-
ing tree is based on a ClustalW amino acid sequence alignment. The bootstrap test
of phylogeny was performed with 10,000 replicates. Most branches are statistically
well supported with confidence values between 95 and 100. The bootstrap values
for the branches separating fungal classes I and III, and insect classes A and B are
below 90 due to the limited number of sequences used in this alignment. Aa, Aedes
aegypti (XP 001662200.1, XP 001651163.1); Af, Aspergillus fumigatus (XP 749322.1,
XP 746604.1, XP 748263.1, XP 752630.1, CAA70736.1, XP 747364.1, XP 754184.1,
XP 755676.1); An, Aspergillus nidulans (XP 662170.1, P30584.2; XP 660127.1;
CBF88263.1; XP 663922.1; XP 659159.1; XP 661971.1; BAE78841.1); Ca, Candida
albicans (XP 717009.1; XP 716433.1; P30573.1; XP717760.1); Dm, Drosophila
melanogaster (AAG22215.3, AAF51798.2); Ms, Manduca sexta (AAL38051.2,
AAX20091.1); Sc, Saccharomyces cerevisiae (NP 014207.1, NP 009594.1,
NP 009579.1); Tc, Tribolium castaneum (NP 001034491.1, NP 001034492.1).
The scale bar represents genetic distance (nucleotide substitutions per site).
class IV enzyme ScCHS3 is responsible both for the formation of
the chitin ring when the bud emerges and for the chitin in the lat-
eral cell wall (see also Fig. 4A). Accordingly, ScCHS3 accounts for
most of the chitin produced in vivo (Shaw et al., 1991). The situa-
tion is more complex in filamentous fungi and hence more difficult
to dissect by genetic approaches. The genome of the filamentous
fungus Aspergillus nidulans, for instance, harbors eight CHS genes,
AnCHSA, AnCHSB, AnCHSC, AnCHSD, AnCHSF, AnCHSG, AnCSMA, and
AnCSMB, which encode class II, III, I, IV, III, VII, V, and VI enzymes,
respectively (Horiuchi, 2009) (see also Figs. 2 and 3). While AnChsB
appears to have crucial roles in hyphal tip growth, AnChsA and
AnChsC have overlapping functions in septum formation and coni-
diation (Motoyama et al., 1996). AnCsmA and AnCsmB perform
compensatory roles in hyphal growth and could be involved in reg-
ulating septal pore formation (Fig. 4B). AnCsmA may be additionally
involved in a signal transduction pathway sensing defects in the cell
wall (Yamada et al., 2005). The functions of the other CHS genes are
largely unknown.
In contrast to fungal genomes, all insect genomes sequenced
so far contain only two CHS genes, which group into class A and
B genes, with the latter group probably constituting the more
ancient form (Muthukrishnan et al., in press) (see Fig. 2). Gene and
761
cDNA sequences are available from various insect orders including
flies (Gagou et al., 2002; Tellam et al., 2000), mosquitoes (Ibrahim
et al., 2000; Zhang and Zhu, 2006), moths (Bolognesi et al., 2005;
Chen et al., 2007; Kumar et al., 2008; Zhu et al., 2002) and bee-
tles (Arakane et al., 2004). The overall structure of CHS genes varies
among different insect species and gene classes with respect to
exon/intron numbers and lengths (Muthukrishnan et al., in press).
Generally, lepidopteran CHS genes appear to be more fragmented
than genes from other insect orders, because they contain a higher
number of shorter exons (Kumar et al., 2008; Zhu et al., 2002). Insect
CHS-A genes are further characterized by the existence of two alter-
nately spliced exons, which are highly conserved between different
insect species (Muthukrishnan et al., in press). Each exon encodes
a 59 aa comprising region consisting of a transmembrane helix
flanked by intra- and extracellular regions. The latter differ from
each other in the presence or absence of a putative N-glycosylation
site. Although it is clear that there is developmental regulation of
alternate exon usage and tissue specific expression of splice vari-
ants, the physiological function remains elusive (Arakane et al.,
2004; Hogenkamp et al., 2005; Zhang et al., 2010a; Zimoch et al.,
2005).
CHS structure and mode of action
CHS enzymes belong to the GT2 family of processive, polymer-
izing glycosyltransferases (Coutinho et al., 2003), which generally
utilize a mechanism where inversion of the anomeric configura-
tion of the sugar donor occurs (Merzendorfer, 2009). GT2 proteins
exhibit a GT-A fold, which is built of two tightly associated ␤/␣/␤
domains forming a continuous central sheet of at least 8 ␤-strands.
The general organization of CHSs has been deduced from a compar-
ison of amino acid sequences mainly from insect and fungal sources
(Merzendorfer, 2006). CHS enzymes can be divided into three dis-
tinguishable regions termed A, B and C domains (Fig. 3). Domain A is
located at the N-terminus and has limited sequence conservation
among different species. In fungal class I–III and VI enzymes the
A domains usually lack transmembrane helices (TMHs), whereas
the A domains of class IV–V and VII enzymes contain 2–3 TMHs. In
class V and some class VI and VII enzymes, the A domain can be pre-
ceded by a myosin motor domain (MMD), which has so far not been
detected in insect but in some mollusc CHSs (Weiss et al., 2006). In
all insect CHS enzymes the A domains contain TMHs with a num-
ber varying between 7 and 10 (Merzendorfer and Zimoch, 2003).
The B domain forms a central catalytic domain which is orien-
tated towards the cytoplasm. It contains several highly conserved
stretches including the GT2 consensus sequences, which include
Walker A and B motifs for binding of the nucleotide moiety (Walker
et al., 1982), the DXD and G(X)4(Y/F)R motifs likely involved in
substrate binding, the GEDRxx(T/S) motif at the acceptor binding
site, and the (Q/R)XXRW motif possibly involved in product bind-
ing (Merzendorfer, 2006). The C domain comprises the C-terminal
part of the CHS and is characterized by the presence of 3 TMHs
in many fungal class IV and VII enzymes, or 7 TMHs in most of
the other CHS enzymes. The TMHs are remarkably conserved with
respect to their sequence, location and spacing (Fig. 3). Particu-
larly striking is the fact that frequently 3–5 of these TMHs form a
cluster (3–5 transmembrane spans, 3–5 TMS) immediately follow-
ing the catalytic domain (Fig. 3). If this cluster consists of 5 TMHs,
which is the case in many fungal and all insect enzymes, two further
TMHs are located closer to the C-terminus. Following the last trans-
membrane helix of the 3–5 TMS cluster a sequence similar to the
(S/T)WGT(R/K) motif originally identified in fungal CHSs is located
at the extracellular site. As mentioned above, all of the insect class A
genes contain two alternative exons, which are located C-terminal
to the 5-TMS cluster and encode the next transmembrane segment
and its flanking sequences (Fig. 3). The sequences of the two alter-
762 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769

Fig. 3. Domain architectures of fungal class I–VII and insect class A and B CHS enzymes. Horizontal bars at the top represent extracellular domains, horizontal bars at the
bottom intracellular domains, and vertical bars transmembrane helices. Red box, alternative exon (AE); blue box, myosin motor domain (MMD); open triangle, QRRRW
signature sequence corresponding to the GT2 consensus sequence (Q/R)xxRW; red filled triangles, (S/T)WGRxT(R/K) motif subsequent to the 3/5TMS cluster; The MMD-,
CHS-A, -B and -D domains are indicated by increasing grey shadings. Scale bar, 500 amino acids.

nate exons are homologous but differ considerably in the regions
flanking the transmembrane segment. This may indicate that the
two different forms of insect class A genes may vary in their abil-
ity to interact with cytosolic and/or extracellular proteins, which
might affect regulation of CHS activity or extrusion of chitin chains.
The presence of a coiled-coil region following the 5-TMS region of
insect class A enzymes may be interpreted in the same direction
(Arakane et al., 2004; Zhu et al., 2002).
Due to the lack of structural data, the molecular mechanism
of chitin synthesis is highly speculative, but a general reaction
scheme can be proposed on the basis of biochemical data avail-
able for CHSs and related glycosyltransferases. The CHS enzyme
transfers the sugar moiety of UDP-GlcNAc to the non-reducing end
of the growing chitin polymer. The polymerization reaction fol-
lows a mechanism, in which the nucleophilic attack by the acceptor
hydroxyl group leads to an inversion of the anomeric carbon of the
donor substrate (Lairson and Withers, 2004). The underlying cat-
alytic mechanism may be similar to inverting glycosyltransferases
from bacteria, for which crystal structures have been determined
(Unligil and Rini, 2000). The proposed reaction cycle involves an
oxocarbenium ion-like transition state and requires a catalytic base,
which deprotonates the incoming nucleophilic acceptor facilitat-
ing SN 2 displacement of the nucleoside-diphosphate. Additionally,
a divalent metal ion may act as a Lewis acid catalyst in the reaction
cycle by stabilization of the leaving nucleoside diphosphate.
How polymerization is initiated in chitin synthesis is still a
matter of debate. Previous suggestions that either soluble or cova-
lently linked primers are required to initiate polymerization remain
unproven (Merz et al., 1999). Priming could also be mediated by
CHS itself or an associated protein, as it is observed for glyco-
gen biosynthesis, which uses glycogenin as the primer (Gibbons
et al., 2002). Another problem arises from chitin stereochem-
istry. Because the polymer is built of GlcNAc residues that are
rotated by 180◦ relative to their neighbors, the repetitive unit is
built from a dimer of GlcNAc (chitobiose). To achieve such alter-
nating orientations of GlcNAc residues in the polymer, the CHS
enzyme has to bind its substrate in an alternating “up/down” con-
figuration. The stereochemical problem of binding GlcNAc in two
different orientations resembles the situation in hyaluronan syn-
thases (HASs), which produce the hyaluronan polymer from two
different monosaccharides, UDP-GlcNAc and UDP-glucuronic acid
(Weigel and DeAngelis, 2007). As class I HASs are related to CHSs,
two binding sites for alternating GlcNAc orientations may occur
also in CHSs, and experiments testing monomeric and dimeric
uridine-derived nucleoside inhibitors on yeast CHS preparations
may support this idea (Yeager and Finney, 2004). The fact that
the catalytic step occurs in the cytosol but the product is found
in the extracellular space leads to another question. How is chitin
translocated across the plasma membrane to reach the extracellu-
lar space? According to one hypothesis, the TMHs of the CHS could
assemble to form a chitin-translocating pore within the membrane
(Cohen, 2001). The fact that some fungal CHSs have only a cluster
of three TMHs (see Fig. 3), a number of which may not suffice to
form a pore of appropriate size, could indicate that these enzymes
depend on multimerization or require additional proteins for chitin
extrusion.
Purification and heterologous expression of CHS enzymes
The current lack of knowledge on CHS structure and function
is mainly due to difficulties in obtaining active soluble CHS prepa-
rations. Several attempts to purify active CHS enzymes have been
made during the last decades using different fungal sources includ-
ing S. cerevisiae, Candida albicans, Mucor rouxii, Coprinopsis cinerea
and Absidio glauca. However, when the active fractions were ana-
lyzed under denaturing conditions, only proteolytic fragments of
the enzyme were detected (Braun and Calderone, 1979; Duran and
Cabib, 1978; Kang et al., 1984; Lending et al., 1991; Machida and
Saito, 1993; Montgomery et al., 1984; Ruiz-Herrera et al., 1980;
Uchida et al., 1996) (see also Table 1). In a more recent study, a class
 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769 763

Fig. 4. Regulatory mechanisms and signaling pathways controlling chitin synthesis in budding yeast, filamentous fungi and insects. Chitin synthesis is controlled at multiple
levels during growth and development. Regulatory mechanisms include transcriptional control of CHS gene expression, posttranscriptional control of CHS processing and
targeting, as well as the control of the activities of the enzymes involved. For details refer to the text. (A) Control of chitin synthesis in S. cerevisiae. Transcriptional control of
CHS2 expression involves the transcription factor Rlm1, which is targeted by the signaling cascades of the cell wall integrity (CWI-P) and the high osmolarity glycerol response
pathways (HOG-P). Furthermore, the exit from the ER is controlled by a signaling cascade named mitotic-exit network (MEN). Control of Chs3 may involve phosphorylation
by Pkc1 facilitating the exit from the trans-Golgi reservoirs (chitosomes). (B) Control of chitin synthesis in A. nidulans. CHSA expression is regulated by the transcription factor
AbaA, which is activated by unidentified upstream regulators. Although the stress responses to high and low osmotic (osm) conditions are well documented, the involved
CWI sensor and signaling cascade remain to be determined. Interestingly, AnRlm1 appears not to be involved in transcriptional regulation of AnCHS genes. (C) Proposed
architecture of a signaling cascade controlling chitin synthesis in the insect integument. Developmental control of CHS1 expression may involve transcription factors such as
Grainy head (Grh), D-Fos and the edysteroid receptor (EcR). The proposed cuticle integrity pathway (CI-P), which may be also initiated by wounding, is based on a signaling
cascade involving yet undefined CI sensors and effectors. It may be assumed that targeting of CHS1 to the plasma membrane via COP II vesicles and transport to the apical
tips of microvilli that are stabilized by actin filaments are further control points.

V CHS (WdChs5) was immunopurified with polyclonal antibodies
from the human pathogenic fungus Wangiella dermatitidis. Again,
the enzyme preparation contained proteolytic CHS fragments when
analyzed under denaturing conditions (Abramczyk and Szaniszlo,
2009). In this regard, a new assay is worth mentioning based on
a chitin-binding protein ChbB from Streptomyces spec., which is
fused with His- and Strep-tags to allow rapid purification of chito-
somes and hence partial purification of fungal CHSs (Herasimenka
et al., 2010). Partial purifications with significant CHS activity in
microsomal fractions have been additionally reported from three
insect sources (Cohen and Casida, 1980; Mayer et al., 1980; Ward
et al., 1991). We have purified MsChs2, an insect class B enzyme,
from the midgut of M. sexta (Maue et al., 2009). The developed
purification protocol in this study included differential centrifuga-
tion, sucrose density centrifugation, solubilization by Triton X-100,
ion exchange chromatography and size exclusion chromatogra-
phy. CHS enrichment was monitored by measuring enzyme activity
and immuno-reactivity with an anti-CHS antibody. After gel per-
meation chromatography the solubilized active enzyme elutes in
a fraction corresponding to a molecular mass between 440 and
670 kDa. Native PAGE yielded a single, immuno-reactive band of
about 520 kDa, thrice the molecular mass of the CHS monomer.
This indicates that indeed an active, oligomeric CHS complex was
purified. Further support comes from electron microscopy, which
yielded negatively stained particles of about 10 nm in diameter that
were still visible after manifold dilutions. As shown in Table 1, some
of the previous studies on fungal CHSs also reported high molecu-
lar mass complexes of about 400–800 kDa, when the enzyme was
analyzed under non-denaturing conditions (Braun and Calderone,
1979; Hardy and Gooday, 1983; Kang et al., 1984; Leal-Morales
et al., 1997; Machida and Saito, 1993; Montgomery et al., 1984;
Ruiz-Herrera et al., 1980). Oligomerization of CHS monomers into
higher molecular weight complexes may therefore be necessary
for biological activity. The significance of this phenomenon in vivo,
however, remains to be determined. As oligomerization has been
reported also for other glycosyltransferases such as cellulose syn-
thases, glucosylceramide synthases and glycogen synthases, it may
reflect a universal property of this class of membrane integral
enzymes (Doblin et al., 2002; Horcajada et al., 2006; Marks et al.,
1999).
In addition to some advances in purifying CHSs from natural
sources, insect and fungal CHSs have been expressed with some
success in heterologous bacterial and yeast systems. As the trans-
membrane segments seem to prevent the production of detectable
amounts of CHS proteins in bacterial systems, we and others tried to
express only the soluble catalytic B domain in E. coli and character-
ized the purified recombinant proteins. Although the expression of
the catalytic domain of BcChs3 from the filamentous fungus Botrytis
cinerea resulted in proteins that were capable of UDP-GlcNAc bind-
ing, chitin synthesis was not detectable indicating that the TMHs
are necessary for chitin formation (Magellan et al., 2010). Simi-
larly, our attempts to express the catalytic domain of the MsChs2
from M. sexta did not reveal catalytically active forms. Nevertheless,
the heterologously expressed catalytic domain was employed for
764 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769
Table 1
Properties of purified CHSs from fungal and insect sources.
M. sexta M. rouxii
Solubilization Triton X100 Digitonin Digitonin
Purification Ion and size exclusion Chitosomal
chromatography preparation
M (kDa) 520 500
Prominent 74, 60, 55 21, 23, 33, 39
fragments (kDa)
Proteolytic Yes Yes
activation
Reference Maue et al. (2009) Ruiz-Herrera et al.
(1980)
the generation of antibodies (Zimoch and Merzendorfer, 2002). A
similar approach for antigen-preparation using the soluble myosin
motor domain (MMD) has also been used to generate antibod-
ies to the class V CHS from W. dermatitidis (Abramczyk et al.,
2009). In contrast to bacterial systems, the production of active CHS
enzymes in yeast systems appears to be more promising. Several
groups reported heterologous expression of fungal CHS enzymes in
yeast. Already Au-Young and Robbins (1990) demonstrated func-
tional expression of CaCHS1 from C. albicans in S. cerevisiae chs1
mutants, and Wang and Szaniszlo (2002) used a similar system
to over-express WdChs3 from W. dermatitidis. Recently, Martínez-
Rucobo et al. (2009) were successful in expressing and purifying
ScChs2 and an N-terminally truncated version in Pichia pastoris.
Both recombinant enzymes had enzymatic properties that were
similar to those reported for the wild-type enzyme from S. cere-
visiae, but they differed in the presence of phosphorylation sites
which evidently influence protein stability. Finally, heterologous
expression of PbCHS3 from the pathogenic fungus Paracoccidioides
brasiliensis in chs3 yeast cells increases CHS activity and chitin
content within the cell wall (Barreto et al., 2010).
EhCh1 from Entamoeba histolytica is the only non-fungal CHS
which was successfully expressed as a functional enzyme in a
chs1 chs3 yeast mutant (Van Dellen et al., 2006). Its specific
activity, which was measured in the membrane fraction, was even
higher in this mutant than that of ScChs3 in wild-type cells, prob-
ably due to its higher expression levels. Interestingly, EhChs1
activity and trafficking was independent of auxiliary proteins that
are required for proper ScChs3 function. In contrast, attempts to
express non-fungal CHS enzymes in bacterial, insect and vertebrate
cell lines failed so far. This includes the production of chimeric
CHS versions replacing different regions of the catalytic domain
of ScCHS3 by corresponding regions of MsChs2 from M. sexta
attempted by us (Meissner, 2010). Possibly, the expression of a
comparably small-sized CHS from the chlorovirus CVK2 in Chlorella
cells will help to obtain sufficient amounts of active enzyme for
structural studies (Kawasaki et al., 2002).
Transcriptional regulation of genes involved in chitin synthesis
In fungi and insects, chitin synthesis is tightly regulated at the
transcriptional level during growth and development. The control
of fungal gene expression is particularly coupled to the regulation of
the cell cycle. A comprehensive study of cell cycle-regulated genes
in S. cerevisiae by microarray hybridization using cells that were
synchronized by three independent methods revealed that the
expression of ScCHS2 was highest in the M-phase and that of ScCHS1
during M/G1 transition, the appropriate times for their functions in
primary septum formation during and cell wall repair after cytoki-
nesis (Lenardon et al., 2010b; Spellman et al., 1998). According to
Choi et al. (1994a), the strongest case for transcriptional regulation
can be made for ScCHS2, because mRNA levels and Chs2 activity vary
periodically during the cell cycle. In contrast, ScChs1 and ScChs3
S. cerevisiae A. glauca C. cinereus W. dermatitidis
Digitonin Digitonin Digitonin Digitonin
Chitin entrapment Copper-chelate Copper-chelate Immunopurification
sepharose Con-A sepharose with anti-MMD
antibodies
570 N.D. 800 N.D.
74, 63 30 63 120, 90
Yes Yes Yes Yes
Kang et al. (1984) Machida and Saito Montgomery et al. Abramczyk and
(1993) (1984) Szaniszlo (2009)
yielded a mixed picture, since both enzymes turned out to be quite
stable after transcription was shut off. Analysis of gene transcrip-
tion during the cell cycle of a C. albicans strain, whose growth can
be synchronized by an ␣-factor induced G1 arrest, showed that the
expression of the class II gene CaCHS1 (orthologous to ScCHS2) and
the class I gene CaCHS8, and to some extent also that of the class IV
gene CaCHS3 (orthologous to ScCHS3) reached highest levels in the
G2 phase shortly before cytokinesis (Côte et al., 2009). In contrast,
the expression of the class I gene CaCHS2 did not correlate with
the cell cycle, but increased like CaCHS3 shortly after induction of
hyphal formation (Munro et al., 1998). In the filamentous fungus
A. nidulans, the expression pattern was investigated for AnCHSA,
AnCHSB, AnCHSC, and AnCHSD using strains expressing lacZ fusions
of the respective genes (Fujiwara et al., 2000). Expression of all
of these genes was detectable to various degrees in hyphae and
increased during conidiation. Transcriptional control of the various
AnCHS genes appears to be independent of AnRlm1, whose homo-
logue regulates ScCHS2 expression in S. cerevisiae (Fujioka et al.,
2007). Instead, the TEA/ATTS transcription factor AbaA was iden-
tified to be a key regulator of asexual development in A. nidulans
(Andrianopoulos and Timberlake, 1994). At least transcription of
AnCHSC, but likely also that of AnCHSA and AnCHSD, is directly acti-
vated by AbaA. Other transcriptional regulators such as BrlA and
MedA may also participate in controlling AnCHSC expression during
asexual development (Park et al., 2003). In contrast, at the onset of
sexual development AnCHSE expression is strongly induced in cleis-
tothecia and hulle cells, while moderately low expression levels are
observed in mature sexual structures (Lee et al., 2005).
Insect CHS1 and CHS2 genes are differentially expressed and
regulated during growth and development. Yet, the underlying reg-
ulatory mechanisms are only poorly understood. While CHS1 genes
are typically expressed over a wider range of developmental stages,
CHS2 genes are not expressed in embryonic or pupal stages, but in
all larval stages and in adults (Muthukrishnan et al., in press). These
developmental differences in CHS1 and CHS2 expression led to the
assumption that insect CHS genes may have specialized functions.
Therefore, we systematically investigated tissue specific expres-
sion in M. sexta (Hogenkamp et al., 2005; Zimoch et al., 2005).
MsCHS1 was found to be expressed in epidermal cells and in the
tracheal system of larvae and pupae, whereas MsCHS2 was detected
only in midgut tissue. These findings suggested that CHS1 enzymes
are involved in the formation of epidermal and tracheal cuticles,
while CHS2 enzymes are involved in midgut peritrophic matrix
formation. Supporting evidence was provided by studies carried
out in T. castaneum, where TcCHS1 is expressed predominantly in
the embryonic and pupal stages, whereas TcCHS2 is prevalent in
the late larval and adult stages (Arakane et al., 2004). Injection of
dsRNA for TcCHS1 to specifically knock down its expression by RNAi
disrupted all types of molts and caused a significant reduction in
total chitin content. In contrast, injection of dsRNA for TcCHS2 did
not affect pupal or adult development, but caused larval starvation.
In line with the assumption that TcCHS2 is involved in peritrophic
 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769
matrix formation, chitin was not detectable in midguts of larvae
treated with dsRNA for TcCHS2, but was abundant when treated
with dsRNA for TcCHS1 (Arakane et al., 2005). Functional special-
ization of Chs1 and CHS2 genes in chitin synthesis for cuticle and
peritrophic matrix formation has been confirmed recently in sev-
eral holometabolous (Chen et al., 2007; Kumar et al., 2008; Zhang
et al., 2010b), and one hemimetabolous insect species (Zhang et al.,
2010a).
In situ hybridization performed with midgut sections from the
yellow fever mosquito Aedes aegypti showed that AaCHS transcripts
(encoding AaChs2) were up-regulated in response to a bloodmeal,
when the peritrophic matrix is formed (Ibrahim et al., 2000). Fur-
thermore, blood feeding also induces transcriptional activity of
AaGFAT1, the gene encoding the glutamine-fructose-6-phosphate
amidotransferase (Kato et al., 2002). Hence, it seems that the con-
trol of chitin biosynthesis in response to environmental changes
involves transcriptional regulation of CHS and GFAT genes in A.
aegypti, but none of the other mosquito genes encoding enzymes
involved in chitin biosynthesis such as UAP, which is constitutively
expressed throughout all life stages (Kato et al., 2005).
Transcriptional regulation of CHS expression during insect
development appears to be complex and so far no factors have
been identified unequivocally to act on cis-regulatory elements of
CHS promoters (Moussian, 2010). As insect molting is hormonally
controlled by ecdysteroids, we postulated that transcriptional con-
trol of CHS genes involves ecdysone-responsive elements in the
upstream regions (Merzendorfer and Zimoch, 2003). Other sug-
gested candidates are the transcription factors D-Fos, and Grainy
head (Grh), which have been shown to trigger the activation of
genes involved in sclerotization and melanization by binding to AP-
1 and Grh elements. Indeed, Krotzkopf verkehrt (kkv), the Drosophila
gene encoding DmCHS1, harbors five potential Grh binding sites
in its first intron, whose role in developmental control of chitin
synthesis, however, remains to be established (Moussian, 2010).
In contrast to A. aegypti, transcriptional control during develop-
ment has been reported for the mummy gene encoding the UAP of
Drosophila flies (Tonning et al., 2006).
Signaling pathways ensuring cell wall and cuticle integrity
Maintenance of cell wall integrity (CWI) during fungal growth
and morphogenesis relies on signaling pathways that induce com-
pensatory alterations upon cell wall stress, which include activation
of chitin synthesis in response to changes in the external environ-
ment provoking cell wall stress. Several signaling pathways have
been identified in fungi that contribute to the maintenance of the
cell wall (Fig. 4A and B). One of them, called the CWI pathway,
appears to be of particular importance, as it orchestrates all com-
pensatory changes to the wall (Levin, 2005). In S. cerevisiae, the
main sensors of this pathway are the membrane integral proteins
ScWsc1 and ScMid2 (Rodicio and Heinisch, 2010). They signal to
the small G protein Rho1 by recruiting the guanine nucleotide
exchange factor Rom2. Rho1 activates different effectors includ-
ing protein kinase C (Pkc1), which triggers a MAP kinase cascade
targeting transcription factor such as ScRlm1 (Garcia et al., 2004).
Again, the ScGFAT1 gene appears to be a target of the CWI path-
way, as it is induced under cell wall stress likely by ScRlm1 binding
to the ScGFAT1 promoter (Bulik et al., 2003; Lagorce et al., 2002;
Levin, 2005). The increase in chitin amounts under cell wall stress
conditions seems also to be a consequence of elevated levels of
ScChs3, which is rapidly mobilized from internal storage com-
partments known as chitosomes and integrated into the plasma
membrane (Valdivia and Schekman, 2003). This mobilization may
be triggered by the upper parts of the CWI pathway. Although
ScChs3 is phosphorylated by Pkc1 in vitro, the precise role in cell
wall stress responses remains unclear (Levin, 2005). Recently, the
765
high osmolarity glycerol response (HOG) pathway, another MAP
kinase cascade initiated by the histidin kinase osmosensor Sln1, has
also been implicated in regulating cell wall remodeling (Bermejo
et al., 2008). It seems that in S. cerevisiae the CWI and HOG path-
ways cooperate in the control of transcriptional responses to cell
wall stress (Rodriguez-Pena et al., 2010). There is also significant
cross-talk to the Ca2+ /calcineurin pathway, which regulates chitin
synthesis in the human pathogenic fungus C. albicans (Lenardon
et al., 2009; Munro et al., 2007). Cell wall stress responses were also
examined in filamentous fungi. Akin to yeast cells, up-regulation of
GFAT1 expression is observed in the cell wall stress response of
Aspergillus niger (Ram et al., 2004), and presumably mediated by an
Rlm1p-like transcription factor (Damveld et al., 2005). The analysis
of AnCHSA, AnCHSB and AnCHSC expression in A. nidulans by North-
ern blots and an sGFP reporter system revealed that AnCHSA and
AnCHSC but not AnCHSB are induced under high salt osmotic stress
(Lee et al., 2004) (see also Fig. 4B). In contrast, the expression of
the class V gene AnCSMA from A. nidulans, which is also produced
in hyphae during vegetative growth periods, is significantly down-
regulated under high osmotic conditions (Takeshita et al., 2002).
The AnCSMA gene is located next to the class VI AnCSMB gene in
the A. nidulans genome, but is transcribed in the opposite direction,
suggesting that transcription is driven by a bidirectional promoter.
Accordingly, the expression of both genes was observed to be up-
regulated under low osmotic conditions (Fig. 4B), and deletion of
one gene altered the expression level of the other gene possibly by
affecting the chromatin structure of the common promoter region
(Takeshita et al., 2002, 2006).
As the integrity of the cuticle is vital to insects an analo-
gous cuticle integrity (CI) pathway akin to the CWI pathway in
fungi may exist, which involves mechano-sensors, signal trans-
duction components and transcription factors that finally regulate
the expression of genes involved in cuticle remodeling (Fig. 4C).
The components of this pathway have yet to be identified in
insects. In line with the CI pathway hypothesis DmCHS1 is strongly
up-regulated in epidermal cells surrounding wounds that were pro-
voked by microinjection needles (Pearson et al., 2009). However,
in this case up-regulation of DmCHS1 (kkv) expression is not medi-
ated by a Grh-like transcription factor as seems to be the case during
Drosophila development. Moreover, the signaling pathway involved
in up-regulation of kkv expression appears to be fundamentally dif-
ferent from that of genes involved in wound healing, such as ddc
and ple genes encoding dopa decarboxylase and tyrosine hydroxy-
lase, respectively. While the latter two genes require the JUN/FOS
and Grh transcription factors to induce the wound response, tran-
scriptional activities of the identified wound enhancer in the kkv
upstream region was not affected by these transcription factors
(Pearson et al., 2009).
Posttranscriptional regulation and intracellular localization
Posttranscriptional regulation of chitin synthesis affects intra-
cellular localization and the enzymatic activities of proteins
involved in chitin biosynthesis. For instance, the activity of GFAT
has been demonstrated to be susceptible to feedback inhibition by
UDP-GlcNAc and controlled by phosphorylation. When DmGFAT1
from D. melanogaster was expressed in yeast cells, the resulting
enzyme was inhibited by UDP-GlcNAc, and stimulated by pro-
tein kinase A (PKA), presumably via a PKA phosphorylation site,
which is also conserved in human and yeast enzymes (Graack et
al., 2001). The PKA phosphorylation site was identified in the C.
albicans CaGFAT as the Ser208 residue (Gabriel et al., 2004). Also
the UDP-GlcNAc pyrophosphorylase (UAP) may be regulated at the
posttranscriptional level by uridine, as this nucleoside was shown
to be an effective inhibitor for the yeast enzyme (Yamamoto et al.,
1980). Analysis of UAP activity in vivo is challenging, as UDP-GlcNAc
766 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769
affects numerous metabolic pathways. Correspondingly, mutations
in the mummy gene encoding DmUAP in Drosophila yielded com-
plex phenotypes, in which next to defects in cuticle formation also
the development of the central nervous system and epithelial orga-
nization are affected (Tonning et al., 2006).
Phosphorylation can also control the intracellular localization
and stability of CHS enzymes. As already mentioned above, ScChs2
synthesizes chitin for the primary septum in S. cerevisiae (Shaw
et al., 1991). ScChs2 is phosphorylated by ScCdk1 at four positions
at the N-terminus and this modification leads to the retention of
ScChs2 in the ER until mitotic kinases are degraded during the
cell cycle (Teh et al., 2009). Dephosphorylation of the N-terminal
domain did not affect CHS activity but resulted in a destabiliza-
tion of ScChs2 (Martínez-Rucobo et al., 2009). In C. albicans, protein
kinases and the establishment of cell polarity have important roles
in the regulation of cell wall composition as well (Blankenship
et al., 2010). This view is supported by the recent finding that
phosphorylation of the class IV enzyme CaChs3 is necessary for
its correct targeting to the plasma membrane at sites of polarized
growth (Lenardon et al., 2010a). The intracellular localization of
ScChs3 has been studied extensively in S. cerevisiae. Fluorescence
microscopic studies have demonstrated that ScChs3 is localized in
smaller amounts at the plasma membrane, in higher amounts in the
membranes of post-Golgi vesicles, and during cell division in a ring-
like structure at the bud neck of small-budded cells (Cabib et al.,
1993). ScChs3 attains its native conformation in the ER, a process
which involves the ER chaperone ScChs7 (Trilla et al., 1999). After
processing of ScChs3 in the ER and Golgi apparatus, which includes
palmitoylation by ScPfa4 and N-glycosylation (Lam et al., 2006;
Santos and Snyder, 1997), it is transported from the trans-Golgi
network to the cell surface. This step requires ScChs5 and ScChs6,
which are part of an exomer coat-complex (Wang et al., 2006). The
formation of the phosphatidylinositol 4-phosphate (PtdIns(4)P)
lipids by the PtdIns(4)P kinase ScPik1 triggers forward transport
of ScChs3 to the plasma membrane, and the signal is eventually
terminated by the phosphoinositide phosphatase ScSac1 (Schorr
et al., 2001). At the bud neck ScChs3 is finally linked to septins
via ScChs4 and ScBni4 (DeMarini et al., 1997). ScBni4 also recruits
the catalytic subunit of protein phosphatase 1 (ScGlc7) to the bud
neck in a temporal and spatially restricted manner. Hence, dephos-
phorylation of a yet unidentified substrate may assist in ScChs3
recruitment (Kozubowski et al., 2003; Larson et al., 2008). ScChs3
is not degraded in vacuoles, but accumulates in specialized vesi-
cles called chitosomes (Ruiz-Herrera et al., 1977), which appear to
act as a trans-Golgi reservoir that is replenished by the endocytotic
turnover of the enzyme (Ziman et al., 1996).
The function of prenylated Chs4 is possibly the most enigmatic
one, as it stimulates ScChs3 activity by an unknown mechanism and
impairs the endocytotic turnover of ScChs3. ScChs4 is required not
only for Chs3 activity but also for the interaction between ScChs3
and ScBni4, which promotes chitin synthesis and proper chitin
localization (DeMarini et al., 1997). ScChs4 is transported to the
plasma membrane independently of ScChs3 (Reyes et al., 2007).
Two groups have reported that Chs4 undergoes prenylation dur-
ing maturation. It is the prenylated form of ScChs4, which seems to
modulate Chs3 activity in vitro and in vivo. The role of the prenyla-
tion as a determinant for the association of Chs4 with membranes,
however, is discussed controversially. Grabinska et al. (2007) sug-
gested that Chs4 prenylation is required for ScChs3 activity but not
for membrane association, whereas Reyes et al. (2007) concluded
that prenylation is required for membrane association but not for
its biological function in chitin biosynthesis. In contrast, we found
that the prenylation of ScChs4 promotes both, chitin synthesis and
membrane association (Meissner et al., 2010).
Unlike ScChs3, intracellular transport of ScChs2 to the bud neck
is under the control of mitotic ScCdk1, whose activity blocks the
exit from the ER. Upon its release, ScChs2 is transported by COPII
vesicles to the bud neck region, where it synthesizes chitin for
the primary septum. In yeast, the exit of mitosis is triggered by
a GTPase-driven signaling cascade called the mitotic-exit network
(MEN). Activation of the MEN promotes mitotic ScCdk1 inactivation
and controls targeting of ScChs2 and other components involved in
cytokinesis, including ScInn1 and ScCyk3 (Jendretzki et al., 2009;
Meitinger et al., 2010).
Another mode of influencing the localization of CHS enzymes
involves N-terminal myosin motor-like domains (MMDs), which
are fused to the CHS domains in some enzymes. MMDs containing
CHSs belong to fungal class V, VI and VII enzymes such as the class V
enzyme from W. dermatitidis (WdChs5; Liu et al., 2004), or the class
V and VI enzymes AnCsmA and AnCsmB from A. nidulans (Takeshita
et al., 2006). Apparently, the MMDs do not exhibit any motor activ-
ity but assist in localizing CHSs to sites of polarized growth by
anchoring them to the actin cytoskeleton (Abramczyk et al., 2009;
Takeshita et al., 2005, 2006). Also in the plant pathogen Ustilago
maydis, the apical localization of the class V CHS UmMcs1 depends
on both, the MMD and F-actin. Long-range movement of UmMcs1
vesicles along microtubules occurs independently from the MMD
(Treitschke et al., 2010). Although insect CHSs generally do not pos-
sess MMDs, they are transported to the apical tips of microvilli,
whose structural cores consist of bundles of actin filaments. It is
tempting to speculate that insect CHSs that are inserted in the
microvillar plasma membrane are drawn along the actin bundles by
separate myosin motor proteins to finally get enriched at the apical
tips. Actually, in S. cerevisiae targeting of ScChs3 to sites of polarized
growth depends on the motor protein ScMyo2 (Santos and Snyder,
1997). Posttranscriptional modification by phosphorylation and
lipidation has not been established for insect CHS enzymes so far.
However, insect CHSs contain putative N-glycosylation sites and
the purified CHS from M. sexta can be stained with sugar staining
dyes (Maue et al., 2009; Merzendorfer and Zimoch, 2003). Although
intracellular trafficking of insect CHSs has not been investigated
systematically, studies of Drosophila mutants defective in compo-
nents of the secretory pathway suggest that proteins involved in
cuticle formation including DmChs1 are transported by COPII vesi-
cles (Norum et al., 2010).
Proteolytic processing and CHS maturation
In various fungal and insect systems, chitin synthesis is stim-
ulated by endopeptidases such as trypsin (see also Table 1). This
observation and the finding of lower molecular mass polypep-
tides in different CHS preparations led to the suggestion that CHS
enzymes exist in two states, either in a zymogenic or an active
state. The biological significance of this phenomenon is still unclear.
In S. cerevisiae, all three CHS enzymes have zymogenic properties.
ScChs1 and ScChs2 have been reported to be activated by trypsin
treatment in vitro (Cabib and Farkas, 1971; Sburlati and Cabib,
1986), but there is no evidence that a proteolytic step plays a role
for regulation of CHS activity in vivo (Roncero, 2002). ScChs3 may
have zymogenic properties as well, but its zymogenicity appears
to depend on UDP-GlcNAc and additional proteins, such as the reg-
ulatory subunit ScChs4 (Choi et al., 1994b; Ono et al., 2000). So
far, no endogenous peptidase has been identified which mediates
zymogen cleavage. The zymogenic properties of yeast ScChs2 and
ScChs3 have been reinvestigated in two recent studies. For ScChs2
it was demonstrated that trypsin acts on a yet unidentified soluble
protease that, once activated, stimulates its activity (Martínez-
Rucobo et al., 2009). On the other hand our results suggest a role for
the CaaX protease ScSte24 in chitin synthesis mediated by ScChs3
(Meissner et al., 2010). ScSte24 is a membrane-integral metallo-
protease residing in the ER where it is involved in CaaX processing
of the yeast mating factor MFa (Tam et al., 1998). Yeast two-hybrid
 H. Merzendorfer / European Journal of Cell Biology 90 (2011) 759–769
studies suggested that ScSte24 interacts with ScChs3, and its inter-
acting domain was mapped to a cytosolic region that immediately
precedes the catalytic domain of ScChs3. A ste24 deletion dis-
plays Calcofluor white (CFW) resistance and decreased chitin levels,
whereas overexpression led to CFW hypersensitivity and increased
chitin levels. Ste24 directly interacts with ScCHS3 but presumably
does not cleave it. Instead, genetic experiments suggest that Chs3
and Ste24 form a complex in the ER that facilitates protease action
on prenylated Chs4, a known activator of Chs3 with a C-terminal
CaaX motif, and that this processing is required for intracellular
transport of Chs3 to the plasma membrane (Meissner et al., 2010).
Akin to fungi, proteolytic activation of chitin synthesis has been
observed in various insect systems. The addition of trypsin to cell-
free extracts obtained from different species such as Diaprepes
abbreviatus, M. sexta, T. castaneum and Stomoxys calcitrans results in
a significant increase in chitin synthesis (Cohen and Casida, 1980;
Mayer et al., 1980; Ward et al., 1991; Zimoch et al., 2005). In Man-
duca, we have shown that trypsin does not directly act on CHS
but on a soluble protein that in turn stimulates chitin synthesis.
This finding shows striking parallels to the detection of a trypsin-
dependent soluble protease that activates ScChs2 mentioned above
(Martínez-Rucobo et al., 2009; Zimoch et al., 2005). In line with the
hypothesis that CHS enzymes are produced as zymogens, dena-
turing gel electrophoresis and immunoblotting of oligomeric CHS
complexes purified from the midgut of M. sexta yielded a dis-
tinct pattern of CHS fragments, which is consistent with two sites
in the CHS monomer that are cleaved during maturation (Maue
et al., 2009). In M. sexta, a chymotrypsin-like peptidase (MsCTLP1)
expressed in the larval midgut was shown to bind to the extracellu-
lar C-terminal domain of MsCHS2 (Broehan et al., 2007). MsCTLP1 is
secreted into the gut lumen and activated by trypsin when the lar-
vae start to feed, a signal which could trigger stimulation of chitin
synthesis (Broehan et al., 2008).
Conclusions
Chitin synthesis in fungi and insects relies on a conserved
biosynthetic machinery which involves soluble enzymes synthe-
sizing the UDP-GlcNAc precursor and a membrane integral CHS
complex polymerizing and extruding chitin. Although insect and
fungal CHSs share only limited sequence homology, the catalytic
domain and the topology of TMHs in the C-terminal domain are
strikingly similar. It can be expected that these similarities will
help to assign structure–function relationships. Recent progress in
expressing active CHSs in heterologous fungal systems and purify-
ing CHSs from natural insect sources furthermore pave the way
towards a mechanistic understanding on the basis of structural
data. Insect CHSs are encoded by only two genes and the derived
enzymes are specialized in the formation of cuticles and PMs. The
degree of functional diversification among CHSs appears to be
higher in fungi. Although fungal CHS genes have partially redun-
dant functions, their activity has been linked to different cellular
processes, such as the formation of the chitin ring at the polarized
growth site, septum formation during cytokinesis, cell wall remod-
eling and repair during vegetative periods of growth, and spore
formation. While the developmental programs that control the
expression of genes involved in chitin synthesis vary in fungi and
insects due to different modes of living, posttranscriptional regula-
tion and processing of the enzymes may follow common principles.
Acknowledgements
The author is grateful to Jürgen Heinisch and Gunnar Broehan
for critically reading the manuscript. This work was supported by
the Deutsche Forschungsgemeinschaft (SFB 431).
767
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