Dynamin-like protein-dependent formation of Woronin
bodies in Saccharomyces cerevisiae upon heterologous
expression of a single protein
Christian Wu
¨rtz, Wolfgang Schliebs, Ralf Erdmann and Hanspeter Rottensteiner
Institut fu
¨r Physiologische Chemie, Ruhr-Universita
¨t Bochum, Germany
The HEX1 protein of Neurospora crassa, identified by
Jedd and Chua [1] and Tenney et al. [2], is the major
component of a class of microbodies limited to euasco-
mycetes and some deuteromycetes, the so-called Woro-
nin body [3,4]. Because of the syncytial growth of
filamentous fungi, wounding of hyphae can lead to a
severe loss of cytoplasm and subcellular organelles, if
the plasma membrane or a nearby septum is not rap-
idly sealed. For this reason, the Woronin body is pres-
ent in filamentous euascomycetes and plugs septal
pores immediately after cells have been damaged [1,2].
In addition to septal pore sealing in cases of injury,
Woronin bodies have also been described as being
required for efficient pathogenesis, survival during
nitrogen starvation [5] and conidiation [6] in various
fungi.
Although it is more than 140 years since the dis-
covery of this very specialized organelle [4], our knowl-
edge of the biogenesis of Woronin bodies remains
incomplete. Electron microscopy studies provided the
first evidence that Woronin bodies are derived from
other microbodies [7]. These findings have been
extended by reports showing that Woronin body
formation is initiated in the vicinity of glyoxysomes
and may proceed through fission from them [8], and
by the demonstration that PEX14 is a key player in
the biogenesis of both glyoxysomes and Woronin
bodies [9]. Furthermore, the presence of a C-terminal
canonical peroxisomal targeting signal type 1 (PTS1) is
required for the proper topogenesis of HEX1 [9] and
allows HEX1 to be imported into peroxisomes upon
heterologous expression in yeast [1]. Peroxisomal
Keywords
filamentous fungi; Neurospora crassa;
peroxisome; protein import; yeast
Correspondence
H. Rottensteiner, Institut fu
¨r Physiologische
Chemie, Abt. Systembiochemie, Ruhr-
Universita
¨t Bochum, D-44780 Bochum,
Germany
Fax: +49 234 321 4266
Tel: +49 234 322 7046
E-mail: hanspeter.rottensteiner@rub.de
(Received 22 January 2008, revised 27
March 2008, accepted 2 April 2008)
doi:10.1111/j.1742-4658.2008.06430.x
Filamentous ascomycetes harbor Woronin bodies and glyoxysomes, two
types of microbodies, within one cell at the same time. The dominant pro-
tein of the Neurospora crassa Woronin body, HEX1, forms a hexagonal
core crystal via oligomerization and evidence has accumulated that Woro-
nin bodies bud off from glyoxysomes. We analyzed whether HEX1 is suffi-
cient to induce Woronin body formation upon heterologous expression in
Saccharomyces cerevisiae, an organism devoid of this specialized organelle.
In wild-type strain BY4742, initial import of HEX1 into existing peroxi-
somes enabled the formation of organelles with a hexagonal crystal. The
observed structures mimicked the shape of genuine Woronin bodies, but
exhibited a lower density and were significantly larger. Double-immuno-
fluorescence analysis revealed that hexagonal HEX1 structures only occa-
sionally co-localized with peroxisomal marker proteins, indicating that the
Woronin-body-like structures are well separated from peroxisomes. In cells
lacking Vps1p and Dnm1p, dynamin-like proteins required for the division
of peroxisomes, the Woronin-body-like organelles remained attached to
peroxisomes. The data indicate that Woronin bodies emerge after the
formation of a HEX1 core crystal within peroxisomes followed by Vps1p-
and Dnm1p-mediated fission.
Abbreviations
PMP, peroxisomal membrane protein; PNS, post-nuclear supernatant; PTS1, peroxisomal targeting signal type 1.
2932 FEBS Journal 275 (2008) 2932–2941 ª2008 The Authors Journal compilation ª2008 FEBS
HEX1 provokes the formation of very small, mem-
brane-bound protein granules that are hexagonal or
spherical [1]. Although intriguing, this observation
points to the existence of additional factors in filamen-
tous fungi that contribute to the formation of mature
and functional Woronin bodies.
Although the transport of HEX1 to Woronin bodies
via microbodies, i.e. peroxisomes, is well known, the
budding of Woronin bodies from microbodies is still
under investigation. Some players in peroxisomal fis-
sion have been identified in recent years, including the
dynamin-like proteins Vps1p [10] and Dnm1p [11]. The
reduction in peroxisome numbers seen in vps1Dand
dnm1Dsingle mutants is even more pronounced in the
absence of both proteins. Most cells possess only a
single peroxisome that is extended and exhibits a num-
ber of constrictions [11]. The Pex11p family of proteins
is also implicated in peroxisome fission, but is thought
to act upstream of the dynamin-like proteins [12–17].
This study is concerned with the topogenesis of
HEX1 using the heterologous Saccharomyces cerevisiae
expression model system.We used diverse cell biologi-
cal and biochemical approaches to scrutinize various
HEX1-expressing strains for the appearance of Woro-
nin-body-like structures. Furthermore, we determined
whether the formation of Woronin bodies from peroxi-
somes shares components of the peroxisomal fission
machinery by examining the fate of HEX1 in a
vps1Ddnm1Ddouble-deletion strain. The results are dis-
cussed in terms of a mechanism for the formation of
Woronin bodies that is largely determined by the
expression of HEX1. The system set-up is likely to be
of use for studying peroxisome fission in a time-
resolved manner.
Results
HEX1 is imported into organelles in S. cerevisiae
in a PTS1-dependent manner
To address whether Woronin body formation depends
on factors specific for filamentous euascomycetes, we
heterologously expressed Neurospora crassa HEX1
cDNA under the control of the constitutive PGK1 pro-
moter in S. cerevisiae strain BY4742. Expression of
HEX1 in BY4742, achieved by the strong constitutive
PGK1 promoter, was verified by western blotting
(Fig. 1A). In line with Jedd and Chua [1], the size of
heterologously expressed HEX1 was the same as that
of endogenous N. crassa HEX1, indicating that HEX1
is correctly synthesized in this yeast strain.
Differential centrifugation confirmed the organellar
localization of HEX1; most HEX1 was found in the
organellar pellet fraction together with the peroxisomal
marker proteins Pex13p and Cta1p, whereas only a
small amount of HEX1 was located in the cytosolic
supernatant (Fig. 1A). HEX1 was also expressed in a
pex5Dstrain, in which PTS1 import is specifically com-
promised because of the absence of the cognate signal
receptor. In this strain, both HEX1 and the peroxi-
somal matrix protein Cta1p were exclusively detected
in the cytosolic fraction, whereas the PMP Pex13p was
still located in the organellar pellet (Fig. 1B). There-
fore, HEX1 is imported into organelles in a PTS1-
dependent manner.
Localization of HEX1 in density gradients
The subcellular distribution of HEX1 was further ana-
lyzed by sucrose density gradient centrifugation. Post-
nuclear supernatant (PNS) obtained from the wild-type
strain BY4742 was subjected to centrifugation at
100 000 gand the resulting pellet was loaded on top of
a sucrose density gradient. As expected, the peroxi-
somal membrane marker Pex13p peaked at a density
of 1.20 gÆcm
)3
and was clearly separated from the
mitochondrial marker Aac2p (1.18 gÆcm
)3
). The perox-
isomal matrix protein Fox3p exhibited a dual distribu-
tion, with one peak corresponding to that of Pex13p
and an additional peak at light fractions (Fig. 1C).
The latter was likely to be caused by ruptured organ-
elles that emerge upon resuspension of the pellet. This
distribution pattern was not altered when a PNS from
BY4742 expressing HEX1 was used. The majority of
HEX1 showed a localization that differed from that
of Fox3p (Fig. 1D), with one peak at a density of
1.23 gÆcm
)3
and one peak at lighter fractions. The dis-
tribution of HEX1 was also distinct from that of
mitochondrial Aac2p, the Golgi ⁄endosome marker
Pep12p and the endoplasmic reticulum marker Kar2p.
When compared with the distribution profile of HEX1
in a density gradient of a N. crassa wild-type strain
(Fig. 1E), it became obvious that in yeast HEX1 did
not sediment to the density of N. crassa Woronin
bodies (1.28 gÆcm
)3
; fraction 5). Thus, in yeast, HEX1
appeared to form Woronin-body-like organelles, but
with a lower density than genuine Woronin bodies.
PTS1-dependent formation of giant Woronin
bodies
The subcellular localization of HEX1 was also analyzed
by immunofluorescence microscopy. Decoration of the
untransformed wild-type strain with anti-HEX1 serum
only led to background staining (Fig. 2A). Analysis of
the HEX1-expressing strain revealed some small
C. Wu
¨rtz et al. Heterologous Woronin body formation
FEBS Journal 275 (2008) 2932–2941 ª2008 The Authors Journal compilation ª2008 FEBS 2933
circular spots typical for peroxisomes. Strikingly, large
hexagonal Woronin-body-like structures with a mean
size of 1.5 ·1.5 lm were also detected upon HEX1
expression (Fig. 2B). Remarkably, typical Woronin
bodies of N. crassa are smaller with an average size of
400–700 nm [18]. In a pex5Dstrain, staining with
anti-HEX1 serum was diffuse (Fig. 2C), thereby verify-
ing the PTS1-dependent import of HEX1 in yeast.
Notably, formation of the hexagonal structures likewise
depended on the peroxisomal import receptor Pex5p.
To examine whether peroxisomal proteins co-localize
with the hexagonal structures, double-immunofluores-
cence staining was carried out in strain BY4742
co-expressing the artificial peroxisomal marker protein
GFP-SKL and HEX1. In a few cells, GFP-SKL showed
a rim-like staining around the hexagonal HEX1 struc-
tures (Fig. 2D, upper). In other cases, GFP-SKL was
located in small dots in vicinity to the hexagonal struc-
tures stained with anti-HEX1 serum. However, most
hexagonal structures did not contain GFP-SKL,
whereas small spots were often double-labeled for
HEX1 and GFP-SKL (Fig. 2D, middle and lower).
Ultrastructure of the hexagonal structures
To corroborate the appearance of hexagonal HEX1
structures in BY4742, this strain was also analyzed by
electron microscopy. The untransformed wild-type
showed a normal distribution and morphology for all
visible organelles (Fig. 3A). In the HEX1-expressing
strain, however, large electron-opaque hexagonal
(Fig. 3B) or rectangular (Fig. 3C) structures were
visible. These clearly harbored a delimiting single
A B
C
D
E
Fig. 1. Subcellular distribution of HEX1 upon heterologous expres-
sion in S. cerevisiae. (A, B) Differential centrifugation. PNS of (A)
BY4742 and BY4742 expressing HEX1, and of (B) the otherwise
isogenic pex5Dgene deletion strain, with or without expressing
HEX1, were separated by centrifugation at 25 000 gfor 20 min into
a supernatant and an organellar pellet fraction. Equal amounts of
each fraction were loaded onto an SDS gel and subjected to
western blot analysis. Distribution of the peroxisomal matrix protein
catalase (Cta1p), the PMP Pex13p and HEX1 was determined with
appropriate antibodies. (C, D) Density gradient centrifugation. The
25 000 gorganellar pellets of (C) BY4742 and (D) BY4742 express-
ing HEX1 were loaded on top of a linear sucrose gradient (30–60%
w⁄w) and subjected to centrifugation at 38 000 gfor 2 h. Fractions
(1 mL) were collected from the bottom (fraction 1) to the top (frac-
tion 27) and assayed by western blot for the distribution of the per-
oxisomal matrix protein Fox3p, PMP Pex13p and HEX1. Aac2p
served as a marker for mitochondria, Kar2p for the ER and Pep12p
for the Golgi ⁄endosome compartment. Densities of the peak frac-
tions of HEX1-containing organelles (1.23 gÆcm
)3
) and peroxisomes
(1.20 gÆcm
)3
) are indicated. (E) Density of Woronin bodies in
N. crassa. For comparison, a 25 000 gorganellar pellet from a
N. crassa wild-type strain was separated on a 30–60% w ⁄w
sucrose gradient and analyzed for the distribution of HEX1 (Woro-
nin bodies), glyoxysomal ICL1 and mitochondrial TIM23. Densities
of the peak fractions of Woronin bodies (1.28 gÆcm
)3
) and glyoxy-
somes (1.20 gÆcm
)3
) are indicated.
Heterologous Woronin body formation C. Wu
¨rtz et al.
2934 FEBS Journal 275 (2008) 2932–2941 ª2008 The Authors Journal compilation ª2008 FEBS
membrane and were therefore designated as giant
Woronin bodies. The size of the hexagonal structures
fitted with the measurements based on the immunoflu-
orescence images. The rectangular structures were up
to 1.5 lm on their short side and up to 7.4 lmon
their long side, and may represent Woronin bodies in
a different orientation with respect to the plane of the
section. The short dimension fits with the measurement
of the en face view of the hexagonal structures. In
some rare cases, peroxisomes with areas of distinct
electron density were denoted (Fig. 3D), probably
representing HEX1-enriched regions that eventually
bud off from the peroxisome.
HEX1 assembles to a crystalline core in
S. cerevisiae
Because the observed Woronin-body-like hexagonal
structures differed from N. crassa Woronin bodies in
density (Fig. 1) and size (Figs 2 and 3), the question
arose as to whether HEX1 is able to form dense crys-
tals in S. cerevisiae. It has been shown previously that
Woronin bodies sediment upon medium speed
centrifugation even if the membrane is removed using
detergent, but this requires proper formation of the
HEX1 core crystal [19]. To this end, differential centri-
fugation at various speeds was conducted, in the
presence or absence of detergent. Examination of a
wild-type PNS revealed increasing amounts of peroxi-
somal marker proteins in the pellet fractions upon
increasing centrifugation speed (Fig. 4). Disintegration
of the organellar membranes by 0.5% Triton X-100
prevented the marker proteins from being sedimented
except for trace amounts in the 15 000 gpellet. A simi-
lar distribution of Cta1p and Pex13p was seen for the
strain expressing HEX1. By contrast, HEX1 was pres-
ent in the 1000 gpellet fraction and, more importantly,
HEX1 was detected in the sediment even after
A
D
B C
Fig. 2. Pex5p-dependent appearance of
giant Woronin bodies upon HEX1 expres-
sion. Yeast strains BY4742 (A),
BY4742 + HEX1 (B) and BY4742pex5D+
HEX1 (C) were analyzed for the localization
of HEX1 by indirect immunofluorescence,
using anti-HEX1 serum in combination with
Alexa Fluor 594-labeled anti-rabbit IgG. In
wild-type cells, large hexagonal structures
resembling Woronin bodies were detected
upon expression of HEX1. (D) Double-immu-
nofluorescence microscopy of BY4742
co-expressing HEX1 and the artificial peroxi-
somal marker GFP-SKL. The three panels
illustrate representative morphological differ-
ences of HEX1-stained organelles. Detection
was achieved with mouse monoclonal anti-
bodies against GFP combined with rabbit
anti-HEX1 serum. The secondary antibodies
used were Alexa Fluor 488-labeled anti-
mouse IgG and Alexa Fluor 594-labeled
anti-rabbit IgG. Bar = 5 lm.
C. Wu
¨rtz et al. Heterologous Woronin body formation
FEBS Journal 275 (2008) 2932–2941 ª2008 The Authors Journal compilation ª2008 FEBS 2935
Triton X-100 treatment, whereas the majority of
Pex13p and Cta1p were detected in the supernatant
fractions. These data indicated that a typical HEX1
crystal core was formed in yeast which is likely to
contain only minor inclusions of peroxisomal matrix
proteins.
Vps1p and Dnm1p: two dynamin-like proteins
involved in fission of peroxisomes and Woronin
bodies
So far, we have been able to show that large Woronin-
body-like structures are formed in S. cerevisiae upon
heterologous expression of N. crassa HEX1. Because
this is supposed to require budding from peroxisomes,
we analyzed whether the typical peroxisomal fission
machinery is also involved in the formation of
Woronin bodies. Key players in peroxisomal fission
are the dynamin-related proteins Vps1p and Dnm1p
[10,11] whose concomitant absence typically results in
the presence of just one giant peroxisome per cell [11].
Thus, if Vps1p and Dnm1p are also required for the
formation of Woronin bodies, HEX1-containing
microbodies should be caught in the act of separating
from peroxisomes in this mutant strain.
The effect of the vps1 dnm1 double-deletion on the
subcellular distribution of HEX1 was first analyzed by
differential centrifugation analysis. HEX1, as well as
Pex13p and Cta1p, were detected in the pellet fraction
(Fig. 5A), although trace amounts of Cta1p also
AB
CD
Fig. 3. Ultrastructure of giant Woronin-body-like organelles and
peroxisomes in BY4742. Cells were grown for 14 h on medium
containing oleic acid as the sole carbon source and processed
for electron microscopy. (A) Typical morphology of a wild-type cell.
(B–D) BY4742 cells expressing HEX1. (B) A Woronin-body-like
structure is viewed from the top, with the typical hexagonal shape
of a N. crassa Woronin body. (C) A giant rectangular Woronin body
is captured from the side. (D) A small Woronin body is still attached
to a peroxisome (*), representing an intermediate of the fission pro-
cess. N, nucleus; M, mitochondria; Ld, lipid droplets; V, vacuole; P,
peroxisomes; Wb, Woronin-body-like structures. (A–C) Bar = 5 lm;
(D) Bar = 2.5 lm.
Fig. 4. Properties of the Woronin body core
crystal. PNS prepared from BY4742- and
BY4742-expressing HEX1 were subjected to
centrifugation at 1000, 5000 and 15 000 g
for 5 min, in the presence or absence of Tri-
ton X-100. The resulting supernatant (S) and
pellet (P) fractions were subjected to SDS-
gel electrophoresis and analyzed by western
blotting for the distribution of HEX1, the per-
oxisomal marker proteins Pex13p and
Cta1p. Disintegration of the membranes by
Triton X-100 changed the distribution of
Pex13p and Cta1p, but not that of HEX1.
Heterologous Woronin body formation C. Wu
¨rtz et al.
2936 FEBS Journal 275 (2008) 2932–2941 ª2008 The Authors Journal compilation ª2008 FEBS