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Molecular Microbiology (2012) 86(4), 894–907 䊏
doi:10.1111/mmi.12026
First published online 20 September 2012
Phosphoribosyl pyrophosphate synthetase, as a suppressor
of the sepH mutation in Aspergillus nidulans, is required for
the proper timing of septation
Guowei Zhong,† Wenfan Wei,† Qi Guan, Zhaofei Ma,
Hua Wei, Xushi Xu, Shizhu Zhang and Ling Lu*
Jiangsu Key Laboratory for Microbes and Functional
Genomics, Jiangsu Engineering and Technology
Research Center for Microbiology, College of Life
Sciences, Nanjing Normal University, Nanjing 210046,
China.
Summary
Timely cytokinesis/septation is essential for hyphal
growth and conidiation in Aspergillus nidulans.
Genetic analyses have identified that A. nidulans has
components of the septum initiation network (SIN)
pathway; one of these, SEPH, is a key player for early
events during cytokinesis. However, little is known
about how the SEPH kinase cascade is regulated by
other components. Here, we demonstrate that the
phosphoribosyl pyrophosphate synthetase family
acts antagonistically against the SIN so that the downregulation of AnPRS family can bypass the requirements of the SIN for septum formation and conidiation.
The transcription defect of the Anprs gene family
accompanied with the reduction of AnPRS activity
causes the formation of hyper-septation as well as the
restoration of septation and conidiation in the absence
of SEPH. Clearly, the timing and positioning of septation is related to AnPRS activity. Moreover, with the
extensive yeast two-hybrid analysis and rescue combination experiments, it demonstrated that AnPRS
members are able to form the heterodimers for functional interacting entities but they appear to contribute
so unequally that Anprs1 mutant display relatively
normal septation, but Anprs2 deletion is lethal. Thus,
compared to in yeast, the AnPRS family may have a
unique regulation mechanism during septation in filamentous fungi.
Accepted 29 August, 2012. *For correspondence. E-mail linglu@
njnu.edu.cn; Tel. (+86) 25 8589 1791; Fax (+86) 25 8589 1526.
†
These authors contributed equally to this study.
© 2012 Blackwell Publishing Ltd
Introduction
Cytokinesis is the process by which a cell splits its cytoplasm, accomplished by the contraction of a contractile
actin ring, to produce two daughter cells. Accordingly,
cytokinesis is the final step in cell division after the nuclear
division of mitosis. For cell division to be successful, chromosome segregation, mitotic exit and cytokinesis must be
executed in this order (Barr and Gruneberg, 2007; Sagona
and Stenmark, 2010). Numerous studies have identified
that mitotic exit requires the activation of the conserved
signalling network, termed the mitotic exit network (MEN),
in budding yeast and the septation initiation network (SIN)
in fission yeast (Krapp et al., 2004; Barr and Gruneberg,
2007; Bedhomme et al., 2008; Brace et al., 2011; Meitinger
et al., 2011). Obviously, to keep the co-ordination of mitosis
and cytokinesis, it is crucial for the cell to carry out these
events in the correct order and at the proper time. Although
organisms of different kingdoms have developed unique
mechanisms to execute cytokinesis, signals that trigger the
onset of cytokinesis are evolutionarily conserved (McCollum and Gould, 2001; Baluska et al., 2006; Kim et al.,
2006; Bedhomme et al., 2008). Thus, the use of simple
eukaryotic microorganisms as a model will let us make
rapid progress in understanding many cellular processes
that are common in mammalian cells and plant cells. Unlike
yeast, the filamentous fungus Aspergillus nidulans contains a mycelium of multinucleate cells that are partitioned
by septa. During the germination in A. nidulans, the conidiospores undergo multiple rounds of nuclear division to
produce eight or 16 nuclei in germlings, but they do not
undergo septation until the cell reaches an appropriate
size/volume, and then forms the first septum near the neck
between spore and germ tube (Harris, 2001). Therefore, as
a whole, inter-compartment development and mitosis in
the mycelium becomes asynchronous (Harris, 2001). In
addition, the septum does not subsequently disappear and
daughter cells remain attached. Based on this characterization, the filamentous fungus A. nidulans is able to endure
more defects in cytokinesis than single-cell yeast. Thus,
A. nidulans is an excellent model organism for allowing
an unambiguous identification of investigating the regulation features of cytokinesis (Gould and Simanis, 1997;
Regulation of PRS on septation in Aspergillus nidulans 895
Fig. 1. Screening of sepH suppressors and the isolation of extragenic mutations.
A. Comparison of colonies of wild-type (LB01) and sepH1 (GQ1) strains grown on YUU agar plates after 2 days at 30°C or 42°C.
B. Representative colony phenotypes in three different categories showing all of them with restored conidiation to some extents at restrictive
temperature 42°C. Strains list from left to right in class I mutation: S1, S3, S5, S7; in class II mutation: S30, S60, S83, S94; in class III
mutation: S45, S47, S82, S110.
C. Phenotypic comparison of colonies of wild type (R153), sepH1 (GQ1), sepH1 and sin110 (S110) and sin110 (Sin110). S110 and Sin110
strains showed remarkably reduced colony size but exhibited conidiation at 42°C compared to the control R153 strain.
Harris, 2001). Previous studies have reported that septum
formation in A. nidulans requires the assembly of a septal
band composed of a dynamic protein complex that is
dependent upon a conserved protein kinase cascade
(Harris, 2001; Kim et al., 2006). The serine/threonine
protein kinase SEPH in A. nidulans, a Cdc7p orthologue
from fission yeast, was first cloned in a screen for
temperature-sensitive cytokinesis mutants. It has been
identified that SEPH plays a central part in the initiation of
septation prior to actin ring formation in A. nidulans (Bruno
et al., 2001; Kim et al., 2006). Notably, downregulation of
the SEPH kinase cascade would abolish septation,
whereas hyper-activation would induce the formation of
multiple septa (Bruno et al., 2001). Thus, SEPH is a positive regulator of the SIN which triggers cytokinesis in
A. nidulans. However, little is known about how the SEPH
kinase cascade is regulated by other components, or
whether there exist any of the negative regulators that act
antagonistically to others in the SIN. To gain insight into
the regulatory mechanisms that underlie septation, 116
mutants that suppressed the defects of sepH in A. nidulans
were isolated by UV mutation in this study. Furthermore,
we found that the defects of the SEPH kinase cascade can
be suppressed by the reduction of phosphoribosyl pyrophosphate (PRPP) synthetase activity.
Results
Isolation of mutants that suppress loss of function of
SEPH during cytokinesis
Previous studies have identified that SEPH from A. nidulans, as a homologue of serine–threonine kinase Cdc7p in
fission yeast, plays an important role during cytokinesis. To
further understand the regulation mechanism in which
SEPH is involved during cytokinesis, we sought the extra-
genic mutations that suppress the loss function of SEPH.
According to a previous finding that septation defects of the
SEPH mutant at a restrictive temperature of 42°C resulted
in failed conidiation, the abolished conidiation will be linked
to septation defects (Liu and Morris, 2000; Liu et al., 2003).
Hence, we used conidiation as an indicator of septation
capability. To visually screen for conidiation, a chartreuse
colour (chaA1) in the GQ1 strain (sepH1, pyrG89, chaA1)
was seen as a colour of conidiospores after mutagenesis
(Fig. 1A). Among the 116 independent mutants that
restored conidiation at a restrictive temperature of 42°C,
three categories were classified based on colony phenotypes at both 30°C and 42°C respectively (Fig. 1B). Mutations in class I, to which most of mutations belonged, had a
robust conidiation capacity at both temperatures. In class
II, the colonies showed almost normal conidiation with a
chartreuse colour at 30°C. Whereas, for an unknown
reason, all mutants of class II at 42°C had light brown
conidiospores. In class III, the colonies displayed similar
colony phenotypes but had a reduced colony size compared to those in class II at 42°C; by contrast, at 30°C, class
III had a significantly reduced growth rate compared to that
of wild type. Theoretically, because 30°C is a permission
temperature for sepH1, the defects at this temperature
must be caused by UV-induced suppressor mutations.
Since a mutation embedding a growth defect phenotype
can be easily cloned, the strain S110 (suppressor of sepH,
No. 110), which has a remarkable growth defect at 30°C,
was selected for further study. To firstly determine whether
the suppressor belonged to the intragenic or the extragenic
mutations for sepH, S110 was crossed to the wild-type
strain R153. Among them (n = 306), two kinds of genotypes in progeny were the same as in the parent strains
GQ1 (sepH1, n = 69) and S110 (sepH1, s110; n = 79), and
the other two types belonged to wild type (n = 84) and a
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
896 G. Zhong et al. 䊏
Fig. 2. sin110 mutation induced a
hyper-septation phenotype and partly restored
septation in the absence of sepH.
The mycelium phenotypes of strains were
stained with DAPI, CFW and actin antibody.
A and B. The sepH1 mutation (GQ1) strain
showed the normal septation at 30°C but
abolished septum completely under the same
restrictive temperature condition 42°C.
C and E. S110 and Sin110 induced premature
septation and slow-growth rate phenotypes at
30°C.
D and F. In the absence of sepH at 42°C,
s110 exhibited septation partly while the
sin110 mutation induced a hyper-septation
phenotype.
G and H. Actin is visualized by indirect
immunofluorescence at sites of septum
formation (arrow) in wild-type hyphae (G). The
sepH and sin110 double mutant restored
normal actin ring (arrow) staining at septation
site (H). All strains were grown on the YUU
liquid medium for 13 h at 42°C. Bars, 10 mm.
new type referred to as sin110 (n = 74). Consequently, the
ratio of the four different types of colonies in the progeny
was about 1:1:1:1. This genetic analysis result suggests
that the mutation site of S110 belongs to the extragenic
mutation, which must be located at a genetic locus other
than the sepH region. To further examine the phenotype
caused by this mutation, the spores of Sin110, S110 and
wild type were inoculated and cultured at 30 and 42°C
separately (Fig. 1C). S110 and Sin110 strains showed
slightly reduced colony size compared to the control R153
strain but both exhibited conidiation at 42°C. Notably, it
seems that Sin110 produced more spores than did S110,
indicating that the mutation in Sin110 caused an antagonistical phenotype compared to sepH mutant.
Sin110 mutation restored septation as well as
conidiation in the absence of SEPH
To further detect whether the restored conidiation was
concomitant with the restoration of septation, we observed
the mycelium phenotypes of GQ1, S110 and Sin110
stained with 4,6-diamidino-2-phenylindole (DAPI) and Calcofluor white (CFW). Upon the germination of the conidium, the formation of septum took place at the ‘neck’ site in
very short germlings from both S110 and Sin110 strains at
30°C (Fig. 2C and E). In comparison, almost no detectable
septa were found in similar size of wild-type germlings. In
addition, three-nuclei basal cell was common to be seen in
germlings in wild-type strain and the average nucleus
number (n = 100) in wild type was 3.15 ⫾ 0.79 in the basal
cell. In comparison, in Sin110 strain, four- or five-nucleus
basal cell was common to be seen (n = 100) and the
average nucleus number was 4.04 ⫾ 0.82 nuclei
(n = 100), indicating disturbed nuclear positioning or the
aberrant formation of delocalized septa. Normally, septum
formation requires the assembly of a septal band, which is
accomplished by the contraction of actin ring. A previous
study has identified SEPH as a central player in the initiation of septation prior to actin ring formation in A. nidulans
(Bruno et al., 2001). To further check if suppression by
Sin110 also restores the actin rings that are absent in the
sepH mutant, an immunostaining experiment for actin was
carried out. As hypothesized, double mutants of sin110 and
sepH were capable of developing a clear actin ring at the
septum site (Fig. 2G and H).
Moreover, as opposed to the sepH1 mutant GQ1 with a
completely stopped septation under the restrictive temperature conditions, Sin110 caused hyper-septation compared to that of S110. When cultured at 42°C, the growthretarded phenotype in Sin110 had been relatively relieved
than that cultured at 30°C but Sin110 mutant still had a
reduced hyphal growth rate with a shorter germling
tube than that of wild type. Consequently, the distance
between septa in Sin110 was 11.76 ⫾ 4.11 (mm) instead of
23.6 ⫾ 3.21 (mm) in wild type under the same cultural
medium (n = 140) so that Sin110 had almost twice number
of septa than wild type did for the same length of hyphae
but there was no significant difference in the number of
nuclei/compartment between them. Thus, according to
the average distance between septa, we concluded that
Sin110 mutant had a hyper-septation phenotype.
Defect of Sin110 can be rescued by phosphoribosyl
pyrophosphate synthetase 1 clone (Anprs1)
To further identify which mutant gene is involved in the
aforementioned suppression of sepH in septation, a complementation test was performed using an A. nidulans
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
Regulation of PRS on septation in Aspergillus nidulans 897
Fig. 3. The functional rescue and recovery of sin110 mutation defects by phosphoribosyl pyrophosphate synthetase 1.
A. Transformant colonies in complementation test grown on YAG agar plates for 3 days after transformed by an A. nidulans genomic DNA
library bearing AMA1 plasmid vector.
B. Colony comparison of wild-type (R153), AMA-Anprs1 (T2) and sin110 (Sin110) strains. Strains were grown on YAG agar plates for 2 days
showing that AMA-Anprs1 can partially restore the sin110 defect at 30°C.
C. The vector map of the rescue plasmid including the AMA vector and Anprs1 (AN6711.4) insert in transformant strain T2.
D and E. T2 mycelia stained with DAPI and CFW displayed the recovery of the septation defect on YAG liquid medium for 13 h. Bars, 10 mm.
genomic DNA library bearing an AMA1 plasmid vector
(Aleksenko and Clutterbuck, 1997). As shown in
Fig. 3A–C, one of the clones that can rescue the defect of
Sin110 was obtained. After sequencing and blasting, this
clone included part of AN6710.4 and the full length of
AN6711.4 located on A. nidulans chromosome I from the
genomic sequence 69087 to 73189 nt, which is referred to
as strain T2. AN6711.4 encodes the putative protein phosphoribosyl pyrophosphate synthetase, called Prs1 in
yeast, which is an important protein phosphatase that is
present in several metabolic pathways (Hove-Jensen,
1988). To further confirm the function of the Prs1 homologue in A. nidulans (AnPRS1) as well as to eliminate the
effect of the fragment in AN6710.4 during the complementation test, the open reading frame (ORF) of the Anprs1
gene was cloned to the AMA1 vector to make a plasmid
called pAMA1-AnPRS1. After transformation to Sin110,
AnPRS1 could consistently rescue the defects of Sin110 to
some extent, suggesting that the expression of the extra
copy of Anprs can partly rescue the defect of Sin110.
Furthermore, using microscopy, as shown in Fig. 3D and E,
relatively normal septation patterns were restored in strain
T2, and even resulted in hyper-septation phenotype at both
30°C and 42°C. According to these complementation data,
it is indicated that the defect of Sin110 is possibly related to
the mutation of the Anprs1 gene. However, after three
independent sequencing assays, the genomic sequence
of a whole gene of Anprs1 in Sin110 mutant was exactly the
same as that in the wild type (Fig. S3). We next wondered
if Sin110 may cause mutation in other members of the
Anprs family. Unexpectedly, no mutation was found in
whole gene regions of Anprs2 or Anprs3 in the Sin110
mutant (Fig. S3). That suggests that the septation defect
suppression of sepH was not due to the mutation of the
Anprs gene family directly, but possibly to other reasons
that affected the function of AnPRS.
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
898 G. Zhong et al. 䊏
Expression and characterization of a functional
GFP–AnPRS1 fusion under the control of the
alcA promoter
To further confirm and test how AnPRS1 functions in
A. nidulans during cytokinesis, we used a conditional strain
in which the Anprs1 gene was under the control of the
inducible/repressible alcA promoter. As shown in Fig. 4A,
homologous integration of the Anprs1 fragment in the
plasmid into the genomic Anprs1 locus generated two
copies of Anprs1: a truncated Anprs1 gene with its own
promoter and a gfp–Anprs1 fusion, called ZGA01, under
the control of the alcA promoter. As shown in Fig. 4B, the
strain ZGA01 had the integration at the desired site. When
ZGA01 was grown on a non-repressing medium (i.e. with
glycerol as the sole carbon source), it displayed a number
of phenotypic similarities to that of the wild type, including
hyphal growth rate, colony size and septation (Fig. 4C).
However, when grown on a repressing medium that contained glucose, ZGA01 induced the phenotype of hyperseptation in germlings or hyphae, accompanied with a
slower growth rate compared to the control strain WJA01
(Fig. 4C, E and F), indicating that this conditional mutant
produced a consistent phenotype with the sin110 mutation.
Meanwhile, GFP–AnPRS1 exhibited a cellular localization
pattern in mature cells. Occasionally, GFP–AnPRS1
appeared at the predicted septation site possibly prior to a
detectable septum formation because in matured septa
there was no detectable GFP–AnPRS1 accumulation
found (Fig. 4D). This suggests that AnPRS1 may function
in the cytosol and septation sites. Moreover, the micrograph examined by transmission electron microscopy
(TEM) showed that, when the expression of AnPRS1 was
turned off, ZGA01 showed the aberrant formation of delocalized septa compared to the wild type (Fig. 4G), which
indicated that AnPRS1 may play an important role in the
timing and positioning of septum formation.
Anprs1 full ORF deletion mutants display normal timing
of cytokinesis
Based on the above growth phenotype of Anprs1 regulated
by the alcA promoter under the repressed condition, we
hypothesized that Anprs1 is not an essential gene in
A. nidulans. We next made a full-length deletion mutant of
Anprs1 (Fig. 5A and B) to further support our observations
of the effect of AnPRS1 on septation in the conditional
strain. Surprisingly, it seemed as if this whole-gene deletion
mutant, referred to as ZGA02, did not show the detectable
septation defect that was observed in the alcA(p)–Anprs1
conditional strain ZGA01 under the repressed condition
(Fig. 5C and D). We wondered whether the truncated
Anprs1 fragment in the conditional strain caused this difference. To test this possibility, a C-terminal truncated
deletion variant of Anprs1 (Anprs1DC), which only had a
truncated fragment of the same length of that in Anprs1 in
the conditional strain ZGA01, was successfully made by
DNA double joint homologue integration of fusion PCR
products (Fig. 5E and F); we refer to this strain as ZGA03.
Perhaps most interestingly, the Anprs1DC mutant caused a
premature and hyper-septation phenotype (Fig. 5G) that
was consistent with strains Sin110 and ZGA01 under the
repressed condition. These data indicate that the existence
of a truncated fragment of AnPRS1 may have the dominant
negative function or have the competitive inhibition for the
AnPRS family under normal circumstances. To test this
hypothesis, we made another double mutant ZGA04 by
crossing Anprs1DC mutant ZGA03 with sepH mutant GQ1.
As a result, Anprs1DC mutant could not completely suppress the defect of sepH in septation. As shown in Fig. S4,
after cultured in liquid medium at 42°C for 20 h, ZGA04 was
unable to form the septum but showed some of more chitin
desposition at the bases of branches than wild type had. It
may indicate that the septation occurred but abolished
during the development of the contractile ring to the mature
septum formation, suggesting Anprs1DC had weaker
ability to suppress sepH mutation than sin110 did.
The defects of Sin110 can be rescued by
AnPRS members
According to all above results, there is a high probability
that when the whole gene of Anprs1 was deleted, other
members of the same family rescued the function.
Genomic information analysis indicated that there were
three members of Anprs in A. nidulans: Anprs1
(AN6711.4), Anprs2 (AN1965.4) and Anprs3 (AN3169.4).
As previously reported, Prs’s predicted function is to catalyse the biosynthesis of PRPP from ribose-5-phosphate
and ATP, the whole process being called PRPP synthetase
activity (Hove-Jensen, 2004). As such we suspect that the
abnormal hyper-septation phenotypes displayed in Anprs1
mutant strains may have some connections with their
PRPP synthetase activity. To prove our hypothesis, biochemical assays to test PRPP synthetase activity were
performed according to standard protocols described in
Experimental procedures. The results indicated that measurable PRPP synthetase activities in extracts from Sin110
and the alcA(p)–Anprs1 conditional strain (ZGA01) under
the repressed condition, as well as from the Anprs1DC
truncated deletion mutant (ZGA03), were substantially
lower than that from the same background wild-type strain
(WJA01) (Fig. 6A). Consequently, the relative AnPRS
enzyme activities in mutant strains ZGA01 and ZGA03
were reduced to 67 ⫾ 3% and 68 ⫾ 5%, respectively, compared to the wild-type strain. Notably, Sin110 caused a
mostly severe defect resulting in only 44 ⫾ 5% of the
retained relative AnPRS enzyme activity compared to the
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
Regulation of PRS on septation in Aspergillus nidulans 899
Fig. 4. The expression and the phenotypic characterization of GFP–AnPRS1 fusion under the conditional promoter.
A. The creation outline of the functional alcA(p)::gfp–Anprs1 strain by highly efficient gene homologous integration strategy.
B. PCR analysis showing the integration of pLB01-Anprs1 into the genome at the original Anprs1 locus in the ZGA01 strain.
C. Colony comparison of alcA(p)::gfp–Anprs1 (ZGA01) on inducing medium MMGPR and on repressing medium YAG after cultured at 37°C
for 2 days.
D. GFP–AnPRS1 was localized in both cytosol and septum sites in conditional strain alcA(p)::gfp–Anprs1 (ZGA01) under the induced
condition. Arrows indicate GFP–AnPRS1 assembled to nearby formed septum. The insets show the enlarged view of the localization band of
GFP–AnPRS1. Bars, 10 mm.
E and F. Septation phenotypic comparison between alcA(p)::gfp–Anprs1 (ZGA01) and wild type (WJA01) was cultured on repressing medium
YAG at 37°C for 10, 12, 15 and 17 h respectively. The nuclei were visualized with DAPI and the septa were stained by Calcofluor white. Bars,
10 mm.
G. Transmission electron micrographs of germlings from wild-type and alcA(p)::gfp–Anprs1 (ZGA01) strains cultured on repressing medium
YAG. Left panel indicates the normal septa while alcA(p)::gfp–Anprs1 (ZGA01, right panel) shows a premature and zigzag septum structure.
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
900 G. Zhong et al. 䊏
Fig. 5. Anprs1DC mutants
caused a premature and
hyper-septation phenotype but
the Anprs1 full-length deletion
displayed the normal timing of
cytokinesis.
A and E. Diagram showing the
deletion strategy for Anprs1 (A)
and the strategy for C-terminus
deletion of Anprs1 (E) based on
the fusion PCR method.
B and F. PCR analysis showed
that the full-length Anprs1 (B)
and the fragment of Anprs1
C-terminal truncated (F)
deletion of Anprs1 were
replaced by AfpyrG at the
original Anprs1 locus in ZGA02
and ZGA03 separately.
C and G (upper panel). Colony
phenotype comparison of wild
type (WJA01) with
alcA(p)::gfp–Anprs1 (ZGA01),
DAnprs1 (ZGA02) and
Anprs1DC (ZGA03) strains on
YAG for 2 days.
D and G (lower panel).
Comparison of septation and
nucleus distribution in hyphal
cells between DAnprs1 (ZGA02)
and wild type, and between
Anprs1DC (ZGA03) and
wild-type strains, all grown in
YAG liquid medium at 37°C for
10 h. The nuclei were
visualized with DAPI and the
septa were stained by
Calcofluor white. The insets
show the high magnification of
the original marked areas. Bars,
10 mm.
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
Regulation of PRS on septation in Aspergillus nidulans 901
Fig. 6. The defects of PRPP synthetase activity, accompanied with the low level of mRNA of AnPRS in Sin110, were able to be rescued by
AnPRS members which could form the heterodimers.
A. The relative AnPRS enzyme activities of wild type (R153), alcA(p)::gfp–Anprs1 (ZGA01), DAnprs1 (ZGA02), Anprs1DC (ZGA03) and sin110
(Sin110). Mycelia were grown in liquid YUU media at 37°C, 220 r.p.m. cultured for 2 days. Data are normalized to a percentage compared to
wild-type strain R153.
B. The relative mRNA levels of Anprs1, Anprs2 and Anprs3, respectively, using real-time RT-PCR assay in the sin110 strain cultured in liquid
YUU media at 37°C 220 r.p.m. cultured for 2 days. The measured quantity of mRNA in each of the treated samples was normalized using CT
values obtained for the actin (AN3696.4) mRNA amplifications running in the same plate.
C. The colony phenotypic comparison of wild type (R153), sin110 (Sin110) and Sin110 transformed by pAMA1-Anprs1, 2 or 3 alone,
respectively, and by an equivalent mixture of pAMA1-Anprs1, 2 and 3.
D. Physical interaction assay among AnPRS1, AnPRS2 and AnPRS3 revealed by yeast two-hybrid system. Full-length cDNA of Anprs2 and
Anprs3 were placed in frame with the DNA binding domain of GAL4 in the pGBKT7 vector while full-length cDNA of Anprs1 and Anprs3 were
constructed in frame into the activation domains in the pGADT7 vector. Protein–protein interactions were detected by the growth of
high-stringency media for selection (SD/-Ade/-His/-Leu/-Trp/X-Gal), and pGADT7-T and pGBKT7-p53 were used as a couple of positive
control for the interaction.
wild type. Accordingly, normal cytokinesis seems to
depend on a normal level of PRPP synthetase activity.
Because the above data have verified that no mutation was
found in Anprs genes in Sin110, we wondered whether the
decreased AnPRS activity in Sin110 was related to the
transcription of the Anprs family. As expected, by real-time
qPCR, three members of the Anprs family in A. nidulans
were dramatically downregulated in Sin110 so that
the mRNA levels of Anprs1, Anprs2 and Anprs3 were
12 ⫾ 8%, 39 ⫾ 5% and 28 ⫾ 9%, respectively, compared
to the wild-type strain WJA01 (Fig. 6B), suggesting that the
mutation of sin110 was mostly related to the reduced
transcription of the Anprs gene family. In response to these
results, we cloned ORFs of Anprs1, Anprs2 and Anprs3
into the AMA1 vector to make the plasmids pAMA1AnPRS1, pAMA1-AnPRS2 and pAMA1-AnPRS3 respectively. As shown in Fig. 6C, the colonies transformed by a
single kind of Anprs plasmid exhibited only partially rescue
of colony size and conidiation. Moreover, it seems that the
rescue efficiency was different in the colonies transformed
by Anprs1 alone, Anprs2 alone or Anprs3 alone. Among
them, Anprs2 induced a stronger rescue than either of
Anprs1 or Anprs3. In comparison, the defects of Sin110 in
growth and septation can be dramatically rescued when
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
902 G. Zhong et al. 䊏
transformed by a mixture of three different Anprs plasmids
at DNA amount ratio 1:1:1, resulting in the transformants
having almost the same colony size and conidiation as the
wild type, indicating that the defects of cytokinesis in
Sin110 can be completely rescued by the expression of the
extra copies of the Anprs family. Thus, AnPRS1 may need
AnPRS2 or AnPRS3 to form an isoenzyme complex to
function. To confirm this hypothesis, next we constructed
the different members of Anprs family into the Gal4 DNA
binding domain and the GAL4 activation domain separately. All combinations (AnPRS1 and AnPRS2; AnPRS1
and AnPRS3; AnPRS2 and AnPRS3) showed a robust
growth in high-stringency media (Fig. 6D), indicating that
the reporter genes (histidine, adenine prototroph and betagalactosidase) could be activated. None of those yeast
cells transfected by single pGBKT7-Anprs or pGADKT7Anprs showed growth under the high-stringency media,
suggesting that none of the bait and prey plasmids had
detectable auto-activation. These results demonstrate that
AnPRS1, AnPRS2 and AnPRS3 are really able to form the
heterodimeric complexes of AnPRS polypeptides.
Discussion
The SIN, which includes the main component Cdc7p and
the GTPase Spg1p, is emerging as a primary regulatory
pathway to control cytokinesis in fission yeast Schizosaccharomyces pombe (Sawin, 2000; McCollum and Gould,
2001; Krapp and Simanis, 2008). In comparison, a functionally similar group of proteins comprise the MEN in
budding yeast Saccharomyces cerevisiae (de Bettignies
and Johnston, 2003; Brace et al., 2011; Meitinger et al.,
2011). Meanwhile, several lines of evidence indicate that
A. nidulans has components of the SIN–MEN pathway
(sepH → sepL → sidB), one of which, sepH, is required for
early events during cytokinesis (Bruno et al., 2001; Kim
et al., 2009; Si et al., 2010). The cytokinesis in fungi can be
viewed as a three-stage process: (i) selection of a division
site, (ii) orderly assembly of protein complexes and (iii)
dynamic events that lead to a constriction of the contractile
ring and to septum construction (Simanis, 2003; Walther
and Wendland, 2003). Nevertheless, in the filamentous
fungus A. nidulans, previous studies have indicated that
actin ring formation, as an initial event of cytokinesis,
occurs after entering mitosis. SEPH, a putative Cdc7p
orthologue, probably functions upstream of actin ring formation during cytokinesis. Thus, it suggests that possibly
SEPH is required during early stages of septation. Further
studies have indicated that SEPH is required for construction of the actin ring, and the deletion of sepH has been
shown to result in a viable strain that fails to septate at any
temperature (Bruno et al., 2001; Sharpless and Harris,
2002; Westfall and Momany, 2002). Therefore, SEPH, as a
major factor in catalysing one of the initial events in cyto-
kinesis, might have to work together with multiple other
protein complexes to regulate cytokinesis. In this study, by
using a combination of forward and reversed genetics
techniques, the results presented here reveal that there
exist protein regulators that act antagonistically towards
components of the SIN in the filamentous fungus A. nidulans. Furthermore, we found that the defects of the SEPH
kinase cascade in septation can be suppressed by the
mutation of sin110 which is caused by the transcription
defects of the Anprs gene family.
Suppressors of SEPH for septum formation in
A. nidulans
Through UV mutagenesis, 116 independent mutants were
obtained that could restore cytokinesis to a certain extent
in the absence of sepH. Among them, three different
classes were delineated based on colony phenotypes, but
all showed septation capabilities to varying extents in
42°C (Fig. 1B). In class I, the mutants had a robust conidiation capacity in the absence of sepH, but at permission
temperature 30°C they did not show the growth defect
phenotype. Thus, they were difficult to clone. In comparison, class II and especially class III mutants could not only
recover cytokinesis but they also displayed a slow-growth
rate phenotype in the presence of sepH. This suggests
that these genes may also have other functions than that
of a negative regulator of cytokinesis; some (if any) of
these functions may belong to the recovery mutation of
sepH (i.e. the intragenic mutation of sepH). To exclude
this possibility, the experiment of back-crossing mutants
with wild type was carried out, and the results clearly
indicate that most mutants had the extragenic mutation
(Fig. S1). Notably, when sepH was defective, the functional mutations of these components could suppress the
failed cytokinesis. Thus, our data clearly provide direct
evidence for the existence of the antagonizing components of the SIN during cytokinesis. Moreover, a prior
suppressor screen has identified smoA and smoB as suppressors of SIN mutations (Kim et al., 2006). Based on
published information combined with ours, we found that
both of sin110 and smoA/smoB mutations caused the
reduced hyphal growth of colony and could suppress
sepH mutations during conidiation, but the hyphal wavy
morphology phenotype, along with the high temperaturesensitive characterization in smoA/smoB mutants, indicated that smoA and smoB could not belong to PRS
family. Solving this puzzle needs the results for further
cloning of smoA and smoB in the future.
The possible relationship between AnPRS and SEPH
It has been reported that in yeast, Prs functions not only in
phospholipid metabolism but also in the MAPK-relied, cell
wall integrity pathway by interacting with major compo© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
Regulation of PRS on septation in Aspergillus nidulans 903
nents of this pathway (Vavassori et al., 2005). Additionally,
Prs3 interacts with the yeast orthologue of GSK3 (glycogen synthase kinase 3) and Rim11, a serine/threonine
kinase involved in several signalling pathways (Kleineidam et al., 2009). Most notably, our data (Figs 3 and 4)
indicate that the decline of AnPRS1 expression suppresses the defect of sepH, and the reduction of PRPP
synthetase activity causes both premature and hyperseptation, which is consistent with the phenotype of overexpression of SEPH in yeast (Bruno et al., 2001). These
findings clearly show that the AnPRS family is involved
in the function of co-regulating cytokinesis with SEPH
in A. nidulans, which has not previously been reported.
We propose a possible explanation of the relationship
between AnPRS and SEPH during cytokinesis in A. nidulans as follows. Because predicted function of AnPRS is
to catalyse the biosynthesis of PRPP from ribose-5phosphate and ATP. However, SEPH, a serine–threonine
protein kinase, also needs ATP to function. Thus, AnPRS1
and SEPH may competitively bind ATP, resulting in the
proper timing of cytokinesis. In addition, there is the possibility that SEPH may act as a protein kinase and
AnPRS1 as a phosphatase to regulate the phosphate and
de-phosphate reactions of the same substrate (not just for
ATP alone). Consequently, this would explain why low
enzyme activity of AnPRS induces the hyper-septation
phenotype as well as the suppression of the SEPH defect.
In addition, in S. pombe, central players of SIN are cascaded kinases of GTPase Spg1p, Cdc7p, Sid1p and
Sid2p (Krapp et al., 2004). Spg1p accumulates in its GTP
bound form, which allows recruitment of the protein
kinase Cdc7p. These kinases and their associated proteins exhibit important function to trigger septation (Sohrmann et al., 1998). To further answer whether the sin110
mutation specifically suppresses SEPH or other SINcascaded kinase mutations in A. nidulans, we deleted
Spg1p homologue in A. nidulans (AN7206.4) in the background of sin110 mutant. As a result, double mutations
showed a robust conidiation (G. Zhong and L. Lu, unpubl.
data) and septation phenotype (Fig. S4). This suggests
Sin110 was capable of suppressing mutations of other
SIN components in addition to SEPH.
The AnPRS family may act as a heterodimer to exert
the biological activity
Phosphoribosyl pyrophosphate synthetase (PRS) catalyses the biosynthesis of PRPP from ribose-5-phosphate
and ATP (Khorana et al., 1958; Roessler et al., 1991). In
the S. cerevisiae genome, there are five unlinked genes
(PRS1–PRS5) capable of encoding the PRS enzyme
(PRS; ATP: D-ribose-5-pyrophosphotransferase; EC
2.7.6.1). None of the PRS genes is essential, but the
contributions of the PRS gene products do not appear to
be equal in S. cerevisiae (Vavassori et al., 2005). Nevertheless, loss of either Prs1 or Prs3 has far-reaching consequences for metabolism, ranging from altered chitin
synthesis and constitutive activation of the cell integrity
pathway to an apparent disturbance in phospholipid
metabolism. Moreover, an extensive yeast two-hybrid
(Y2H) analysis and deletion combination experiments
demonstrated that viable minimal subunits exist as two
interacting functional entities (Prs1/Prs3, Prs2/Prs5 or
Prs4/Prs5 in wild type) which seem to be capable of
compensating for each other because, in the absence of
one entity or one of its components, the yeast cells still
survive (Hove-Jensen, 2004). This clearly suggests that
Prs activity in S. cerevisiae is carried out by heterodimeric
complexes of Prs polypeptides. Based on homologue
analysis, we found three predicted PRS genes in A. nidulans: Anprs1 (AN6711.4), Anprs2 (AN3169.4) and Anprs3
(AN1965.4). According to the protein sequence analysis
of identity and homology in alignment by DNAStar software, AnPRS1 is a putative homologue of yeast Prs1
whereas AnPRS3 is most likely a homologue of Prs5
(identity number 45.54%) and not of Prs3 (identity number
36.45%) as expected, in yeast. We hypothesized that
AnPRS2 may have an important comprehensive function
as a partner of Prs2, Prs3 and Prs4 in yeast. In fact, the
deletion of Anprs2 in A. nidulans resulted in cell death,
indicating that AnPRS2 may be a central subunit functioning as an interactive AnPRS heterodimer to exert its
biology activity which is completely different from the
homologue in budding yeast (Fig. S2). Nevertheless, our
data (Fig. 5C and D) indicate that the whole Anprs1 gene
deletion mutant ZGA02 did not show any detectable
defects while a C-terminal truncated deletion variant of
Anprs1 (Anprs1DC) ZGA03 caused defects in septation
and AnPRS enzyme activity. Moreover, Anprs1DC had
weaker ability to suppress sepH mutation than Sin110
did (Fig. S4) possibly due to the difference between
Anprs1DC and sin110 mutants in retained AnPRS enzyme
activity (Fig. 6). This suggests that the existence of a
truncated fragment of Anprs1 may disturb the normal
function of Anprs2 or Anprs3, which may function similarly
to a dominant-negative mutant. Furthermore, Y2H experimental data in Fig. 6 demonstrate that AnPRS members
in A. nidulans are able to form the heterodimers for interacting functional entities AnPRS1/AnPRS2, AnPRS2/
AnPRS3 and AnPRS1/AnPRS3. Perhaps, in the absence
of AnPRS1, functional AnPRS2/AnPRS3 may be capable
of compensating for the function of the AnPRS1 complex
with AnPRS2 or AnPRS3. In contrast, when a truncated
Anprs1DC existed, it is able to induce a competitive inhibition with other members of AnPRS to form a functional
AnPRS complex. Moreover, we found that the contributions of the Anprs gene products do not appear to be
equal in A. nidulans, such that Anprs1 was not essential
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
904 G. Zhong et al. 䊏
(Fig. 5C and D), but deletion of Anprs2 resulted in cell
death (Fig. S2), indicating that the AnPRS family may
have a unique mechanism in filamentous fungi that is
completely different from single-cell yeast. Future studies
on AnPRS function as a heterodimer should clarify the
interaction details in the AnPRS family.
Experimental procedures
Strains, media, culture conditions, plasmids and
transformation
A list of A. nidulans strains used in this study is provided in
Supporting information (Table S1). The media MM (minimal
media), YAG (yeast + agar + glucose media), YUU (YAG +
uridine + uracil), YUUK (YUU + KCl) and MMGPR (MM +
glycerol + pyrodoxine + riboflavin) are described in previous
references (Kafer, 1977; Wang et al., 2009). MMGTPR:
MMGPR with 6.25 mM threonine. Growth conditions, crosses
and induction conditions for alcA(p)-driven expression were as
previously described (Wang et al., 2006). Expression of
tagged genes under the control of the alcA promoter was
regulated by different carbon sources: repression on glucose,
derepression on glycerol and induction on threonine. Standard
DNA transformation procedures were used for A. nidulans
(Osmani et al., 1988; May, 1989). The plasmids used are listed
in Table S3.
Mutagenesis and screening for septation revertants
Strain GQ1 bearing the chaA1 mutation was used for mutagenesis. Approximately 107 conidia were suspended in 20 ml
of sterile distilled water. They were irradiated with ultraviolet
light at a dosage of 8000 mW cm-2 for 65 s with agitation
using CL-1000 (Ultra-Violet Products, Unit1). The irradiation
rendered 10% viability. Mutagenized spores were plated on
the YUU medium. After being incubated at 42°C for 3 days,
the colonies showing the chartreuse colour were picked as
conidiating revertant candidates for further analysis.
Complementation test of the sin110 mutation
The complement test of sin110 was performed using the
pRG3–AMA–NotI genomic DNA library as follows (Yelton
et al., 1984; Osherov et al., 2000). Transformants were
selected for restoration of pyrimidine prototrophy on the YAG
medium. The isolates showing the rescue phenotypes of
sin110 defects were selected for further study. Next, plasmids
were extracted from these rescued Aspergillus clones and
then transformed to competent Escherichia coli as previously
described (Rasmussen et al., 1990). After purifying the plasmids from the positive clones, we retransformed these purified plasmids separately to the Sin110 strain to confirm the
rescued phenotypes. Then, the genomic insert in pRG3–
AMA–NotI was end-sequenced using vector-specific primers
and blasted by the A. nidulans genome database (Aspergillus
Sequencing Project, Broad Institute of MIT and Harvard).
Tagging of AnPRS1 with GFP
To generate an alcA(p)–gfp–Anprs1 fusion construct, a
1133 bp fragment of Anprs1 was amplified from TN02A7
genomic DNA with primer Rec-Anprs1-5′ (NotI site included)
and primer Rec-Anprs1-3′ (XbaI site included) (Table S2).
The 1133 bp amplified DNA fragment was cloned into the
corresponding sites of pLB01, yielding pLB-Anprs1 5′ (Liu
et al., 2003). This plasmid was transformed into TN02A7.
Homologous recombination of this plasmid into the Anprs1
locus should result in an N-terminal GFP fusion of the entire
Anprs1 gene under control of the alcA promoter and a fragment of Anprs1 under its own promoter. The transformant,
which formed the normal colonies under the inducing condition but showed slow-growth defects at 30°C under the
repressing conditions, was subjected to diagnosis PCR
analysis using a forward primer (GFP upstream) designed to
recognize the gfp sequence, and a reverse primer (Anprs1
downstream) designed to recognize the Anprs1-3′ sequence.
Transmission electron microscopy
Transmission electron microscopy examination was carried
out mainly as described in previous research (Horiuchi et al.,
1999; Ichinomiya et al., 2005). In brief, for sample preparation,
germlings grown on YAG plates for 10 h were fixed firstly for
5 h in 0.5% glutaraldehyde with 0.1 M phosphate buffer
(pH 7.0), and then washed by 0.1 M phosphate buffer
(pH 7.0). Next the sample was fixed in a secondary fixation
including 2% osmium tetroxide for 2 h in 0.1 M phosphate
buffer. After dehydration with a graded ethanol series, specimens were embedded in epoxy resin. The sections, cut on an
ultramicrotome with a glass knife, were stained with uranyl
acetate and lead citrate and observed by TEM (Hitachi
H-7650).
Constructions of gene replacement strains
A strain containing the Anprs1 null mutation was created by
double joint PCR (Yu et al., 2004). The Aspergillus fumigatus
pyrG gene in plasmid pXDRFP4 was used as a selectable
nutritional marker for fungal transformation. The linearized
DNA fragment 1 which included a sequence of about 563 bp
that corresponded to the regions immediately upstream of the
Anprs1 start codon was amplified with the primers 5′Anprs1For and 5′Anprs1-Rev+Tail (Table S2). Linearized DNA
fragment 2 including a sequence of about 801 bp that corresponded to the regions immediately downstream of the
Anprs1 stop codon was amplified with primers 3′Anprs1For+Tail and 3′Anprs1-Rev (Table S2). Lastly, purified linearized DNA fragments 1 and 2 plus the pyrG gene were mixed
and used in a fusion PCR with primers Nested-Forward and
Nested-Reverse. The final fusion PCR products were purified
and used to transform A. nidulans strains TN02A7. A similar
strategy was used to construct the truncated strain by using
primers 5′For-Anprs1 and 5′Rev-Anprs1 for the 5′ region,
3′For-Anprs1 and 3′Rev-Anprs1 for the 3′ region, and 5′NestAnprs1 and 3′Nest-Anprs1 for the fusion product. The final
2190 bp Anprs1 5′-AfpyrG-Anprs1 cassette was purified and
used to transform A. nidulans strains TN02A7. The Anprs2
deletion strain and sin110/DspgA double mutant strain were
constructed by using the similar strategy and transformed to
TN02A7 and Sin110-1 strains respectively. Oligonucleotides
used in this study are listed in Table S2.
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907
Regulation of PRS on septation in Aspergillus nidulans 905
Microscopy and image processing
Several sterile glass coverslips were placed on the bottom of
Petri dishes, and gently overlaid with liquid media containing
conidia. Strains were grown on the coverslips at related temperature prior to observation under microscope. The GFP–
AnPRS1 signal was observed in live cells by placing the
coverslips on a glass slide. DNA and chitin were stained using
DAPI and CFW (Sigma Aldrich, St Louis), respectively, after
the cells had been fixed with 4% paraformaldehyde (Polyscience, Warrington, PA) (Harris et al., 1994). For immunofluorescent detection of actin, the growing hyphae were fixed
in PBS (pH 7.4) with 4% paraformaldehyde at room temperature for 30 min. After three washes in PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min, washed three
times with PBS and blocked in blocking buffer (1% bovine
serum albumin in PBS) at room temperature for 30 min. Cells
were then stained with antiactin mouse monoclonal (MP,
1:200) and Alexa Fluor 546 rabbit antiactin mouse IgG (Invitrogen, 1:400) as primary and secondary antibodies respectively. Differential interference contrast (DIC) images of the
cells were collected with a Zeiss Axio Imager A1 microscope
(Zeiss, Jena, Germany). These images were then collected
and analysed with a Sensicam QE cooled digital camera
system (Cooke Corporation, Germany) with MetaMorph/
MetaFluor combination software package (Universal Imaging,
West Chester, PA) and the results were assembled in Adobe
Photoshop 7.0 (Adobe, San Jose, CA).
measuring buffer (KH2PO4/K2HPO4 at pH 7.5, 5 mM MgCl2,
3.75 mM R5P, 2 mM ATP, 3.75 mM phosphoenolpyruvate,
0.2 mM NADH, 1.5 U myokinase, 3 U pyruvate kinase and
1.5 U lactate dehydrogenase). The oxidation of NADH was
measured by monitoring absorption at 340 nm using a Multiskan Spectrum (Thermo Electron Corporation, Waltham, MA,
USA) at room temperature. The specific activity of AnPRS is
expressed as mmol min-1 (mg of NADH oxidized)-1, using the
molar extinction coefficient of 6220 M-1 for NADH. The
soluble protein content of the supernatant was determined
using a dye binding assay (Bradford, 1976).
Interaction analysis by yeast two-hybrid analysis
Saccharomyces cerevisiae strain AH109 (Clontech, Palo
Alto, CA) was used as the host for the two-hybrid interaction
experiments. The full length of cDNA of Anprs2 (AN1965.4)
and Anprs3 (AN3169.4) was placed in frame with the DNA
binding domain of GAL4 by polymerase chain reaction (PCR)
amplification from the UniZAP A. nidulans cDNA library, and
subcloned into the pGBKT7 vector (Clotech, Palo Alto, CA).
Full-length cDNA of Anprs1 and Anprs3 were used for the
activation domains in the pGADT7 vector. The histidine and
adenine prototrophy and b-galactosidase were performed
according to the Clontech Yeast Protocols Handbook
(Clotech, Palo Alto, CA).
Acknowledgements
RNA isolation and quantitative RT-PCR analysis
The mycelia were cultured for 2 days in related media, and
were then pulverized to a fine powder in the presence of
liquid nitrogen. Total RNA was isolated using Trizol (Invitrogen, 15596-025) following manufactory instruction. One
hundred milligrams of mycelia per sample was used as the
starting material for the determination of total RNA. Reverse
transcription polymerase chain reaction (RT-PCR) was
carried out using SuperScript™III First Strand Synthesis
System (Invitrogen, 18080-051), and then cDNA was used for
real-time analysis. For real-time reverse transcription quantitative PCR (RT-qPCR), independent assays were performed
using SYBR Premix Ex TaqTM (TaKaRa, DRR041A) with
three biological replicates, and expression levels were normalized to actin mRNA level. The 2DCT method was used to
determine the change in expression.
This work was financially supported by the National Natural
Science Foundation of China to L. L. (NSFC31070031,
30770031) and Natural Science Foundation of the Jiangsu
Higher Education Institutions of China (Grant No.
11KJA180005) and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the
Research and Innovation Project for College Graduates of
Jiangsu Province (CXZZ11_0885). A. nidulans strain TN02A7
was a gift of B.R. Oakley (Ohio State University, Columbus,
Ohio); A. nidulans strain AJM68 was a gift of Christopher J.
Staiger (Purdue University, West Lafayette); Plasmid pLB01
was a gift from Dr Bo Liu (University of California, Davis);
Plasmid pXDRFP4 and pRG3–AMA–NotI genomic DNA
library and UniZAP A. nidulans cDNA library were from FGSC
(http://www.fgsc.net).
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Supporting information
Additional supporting information may be found in the online
version of this article.
© 2012 Blackwell Publishing Ltd, Molecular Microbiology, 86, 894–907