Integrity of a Zn finger-like domain in SamB is crucial for morphogenesis in ascomycetous fungi.

Abstract

Genetic features determine the site of polarized growth in filamentous fungi and lead to hyphal tip extension or subapical branching. We have isolated the samB gene (suppressor of anucleate metulae) of Aspergillus nidulans which encodes a 66 kDa protein carrying an atypical Cys4 and an additional Cys2/His/Cys Zn finger motif at the carboxy-terminus. Such novel Zn finger-like domains have recently been found in several other developmental regulators in organisms ranging from yeast to man. Deletion of this domain at the carboxy-terminus of SamB led to premature hyphal ramification, mislocalization of septa and suppression of the asporogenous phenotype of the developmental mutant aps (anucleate primary sterigmata). A DeltasamB deletion strain displayed an identical phenotype. A homologous gene in Saccharomyces cerevisiae was also characterized whose deletion resulted in a multi-budding phenotype; thus it was named MUB1. An underlying common mechanism for both genes in determination of the onset of polarized growth and its links to other cellular developmental processes is discussed.

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The EMBO Journal Vol.17 No.1 pp.204–214, 1998
Integrity of a Zn finger-like domain in SamB is
crucial for morphogenesis in ascomycetous fungi
Miriam Kru ¨ger and Reinhard Fischer1
Philipps Universita ¨t Marburg, Laboratorium fu ¨r Mikrobiologie and
Max-Planck-Institut fu ¨r terrestrische Mikrobiologie,
Karl-von-Frisch-Str., D-35043 Marburg, Germany
1Corresponding author
e-mail: FischerR@Mailer.Uni-Marburg.de
Genetic features determine the site of polarized growth
in filamentous fungi and lead to hyphal tip extension
or subapical branching. We have isolated the samB
gene (suppressor of anucleate metulae) of Aspergillus
nidulans which encodes a 66 kDa protein carrying an
atypical Cys4and an additional Cys2/His/Cys Zn finger
motif at the carboxy-terminus. Such novel Zn finger-
like domains have recently been found in several other
developmental regulators in organisms ranging from
yeast to man. Deletion of this domain at the carboxy-
terminus of SamB led to premature hyphal ramifica-
tion, mislocalization of septa and suppression of the
asporogenous phenotype of the developmental mutant
aps (anucleate primary sterigmata). A ∆samB deletion
strain displayed an identical phenotype. A homologous
gene in Saccharomyces cerevisiae was also character-
ized whose deletion resulted in a multi-budding pheno-
type; thus it was named MUB1. An underlying common
mechanism for both genes in determination of the
onset of polarized growth and its links to other cellular
developmental processes is discussed.
Keywords: Aspergillus nidulans/budding/development/
filamentous fungi/Zn finger
Introduction
Fungi are able to grow by two alternative mechanisms,
budding and filamentous growth. Cell growth is achieved
accordingly by isotropic expansion of the cell or polarized
tip extension. The two growth forms contribute to different
extents to fungal morphogenesis in filamentous and in
yeast-like organisms.
In filamentous species, polarized growth dominates
during vegetative stages of the life cycle but isotropic
expansion is important for the development of specialized
cells for reproduction. Polarized growth requires a con-
tinuous supply of cell wall material at the hyphal apex,
which is achieved by an active tipward vesicle transport
(Gow, 1994). This organelle movement requires an intact
actin cytoskeleton in combination with force-generating
motor proteins like myosin (McGoldrick et al., 1995).
Morphogenesis of filaments is normally accompanied by
septation and subapical branching. In the ascomycetous
fungus Aspergillus nidulans, the first septum is formed
once a germinating hypha has reached a certain length
and nuclei have completed the third round of mitoses
204
© Oxford University Press
(Wolkow et al., 1996) leading to eight nuclei. Septum
formation occurs in the rear of the germtube close to the
spore, resulting in 5–6 nuclei in the tip compartment and
2–3 in the spore. The molecular biology underlying these
processes has just begun to be investigated. Several genes
have been described which affect tip growth, branch
formation or septation (Harris and Hamer, 1995; Harris
et al., 1997).
In yeast, cell volume increases by isotropic growth
which is followed by a temporary switch to polarized tip
extension during budding (Cid et al., 1995). In Saccharo-
myces cerevisiae, bud site selection depends on the site
of the previous bud (Chant, 1994). In haploid cells,
daughter cells bud off near the site of the previous bud
(axial budding) whereas in diploid cells daughter cells
bud off alternately at both ends of the cell (bipolar
budding). Axial and bipolar budding share some proteins
but also require some specific components (Park et al.,
1997). Structural genes, like chitin synthase 3 (Santos and
Snyder, 1997), are as important as regulatory genes, like
protein phosphatase 2 PPH22 (Evans and Stark, 1997).
Three classes of proteins have been described which are
involved in the establishment of polarity and thus bud site
selection. One class of proteins localizes to the bud site
and remains there during bud emergence and growth, e.g.
Bem1 (Chenevert et al., 1992), Myo2 (Lillie and Brown,
1994) or Cdc42 (Ziman et al., 1993). Other proteins, like
Cdc3 or Cdc11, assemble at the bud site but remain at
the budding neck as the daughter cell grows (Ford and
Pringle, 1991; Kim et al., 1991). A third class of proteins
is involved in the onset of polarity without an asymmetric
subcellular localization. It is not surprising that some of the
regulatory genes coding for these proteins are conserved in
organisms ranging from bacteria to man, because polarized
growth is essential in all developing cells (Chant, 1994).
A switch from filamentous growth to yeast-like budding
is found in fungi like A.nidulans during maturation of the
asexualreproductive structure,
(Timberlake, 1990, 1991). From a foot cell, a stalk
develops by apical extension until it swells to a globose
vesicle at the tip. In a budding-like process, primary
sterigmata (metulae), secondary sterigmata (phialides) and
conidiospores are produced. Besides the coordination of
the two growth forms, nuclear distribution is essential for
development. Nuclei migrate from the vesicle into the
metulae, which remain uninucleate throughout further
steps of development. All subsequent cell types are also
uninucleate. Two mutants have been described, apsA and
apsB (anucleate primary sterigmata), in which metulae
fail to differentiate further because of a misdistribution of
nuclei. The same mutations have only a slight effect on
hyphal growth. Nuclei are clustered in the multinucleate
hyphal cells of A.nidulans rather than evenly distributed
like in the wild-type (Clutterbuck, 1994; Fischer and
theconidiophore

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Morphogenesis in fungi
Timberlake, 1995; Cai et al., 1996). The apsA gene was
analyzed at a molecular level. It encodes a 183 kDa
coiled-coil protein which localizes at the cytoplasmic
membrane. A pleckstrin homology (PH) domain at the
carboxy-terminus of the protein suggests a role in signal
transductionleadingtopositioningofthenuclei(Suelmann
et al., 1997). The apsB gene was also cloned and
sequenced. This gene encodes a 121 kDa coiled-coil
protein (R.Suelmann, N.Sievers, D.Galetzka, L.Robertson,
W.E.TimberlakeandR.Fischer,manuscriptinpreparation).
To elucidate the molecular functions of apsA and apsB, a
suppressoranalysiswasinitiatedwhichledtothediscovery
of at least two extragenic suppressors, samA and samB
(Kru ¨ger and Fischer, 1996). While samA was dominant,
the recessive locus samB, which was located on chromo-
some VIII, could be complemented by transformation with
a wild-type library. In this study, samB was cloned,
sequenced and the gene product localized subcellularly.
We present evidence that samB encodes a protein, which
is involved in regulation of morphogenesis rather than in
nuclear distribution, and that its function is conserved in
yeast and filamentous ascomycetes.
Results
Molecular cloning of samB
The lack of green spores on conidophores of A.nidulans
apsA mutant strains leads to brown colonies easily distin-
guishable from green wild-type colonies. In the suppressor
strain SMI12 (apsA–; samB–), conidia production was
restored due to the mutation of samB. The samB mutation
had little effect on the phenotype in apsA?strains which
were not suitable for isolation of the gene by comple-
mentation. Therefore, the double mutant was used for
cloning the samB gene. After successful complementation
of the recessive mutation with the wild-type gene, an
apsA-like phenotype was expected, which would appear
inbrowncolonies amonggreennon-complementedstrains.
In one co-transformation experiment using a chromosome
VIII-specific library and the self-replicating plasmid
pAD4ARp1, five strains with an apsA-like phenotype
were isolated among 2000 transformants. After colony
purification of the original transformant, the comple-
menting hybrid plasmid was isolated. This plasmid com-
plemented the samB defect with a high frequency,
indicating that it contained the entire samB gene. Because
severe rearrangements of the genomic DNA inserted into
the pAD4ARp1 plasmid can occur, we isolated a cosmid
from the same library by hybridization with restriction
fragmentsfrom genomic
pAD4ARp1. We identified one cosmid which mapped to
chromosome VIII, which is in agreement with the classical
mapping data for samB. This cosmid (SL10E10) comple-
mented the mutation with high frequency. Using gel-
isolated restriction fragments obtained after digestion of
the cosmid with EcoRI, the smallest complementing piece
of 4.1 kb was identified in a co-transformation experiment
with the pyr4- (pyr4 complements the pyrG mutation)
containing plasmid pRG1. Within this 4.1 kb region, an
internal EcoRV restriction fragment hybridized to a 2.7 kb
transcript isolated from hyphae. The genomic DNA frag-
ment was cloned into pBluescript (pMir25) and used for
sequence analysis (Figure 1).
DNA ratherthanfrom
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Sequence of samB
The 4.1 kb insert of pMir25 was sequenced on both
strands. To investigate whether the samB gene harbors
any introns, the 4.1 kb fragment was used to isolate six
corresponding cDNAs from a λgt10 cDNA library (kindly
provided by G.May, Houston, TX). Sequence comparisons
of three of the cDNAs with the sequence of the genomic
fragment revealed no intron in the entire coding region;
the cDNAs started at positions 633, 658 and 739. In the
600 bp upstream of the putative transcriptional start sites,
three CAAT boxes and two TATAAA-like boxes were
found. In addition, sequences preceding the start points
contain CT-rich stretches. At the 3? end of the cDNAs,
polyadenylation sites 0.6 kb downstream of the stop codon
at positions 3273, 3276 and 3282 were detected. Open
reading frame (ORF) analysis revealed one region encod-
ing a polypeptide of 590 amino acids (Figure 1). Transla-
tion started with a methionine at base pair 865. In the
232 bp region between the initiation of the longest cDNA
and the first ATG, no further ATG-initiated ORFs were
detected. The N-terminal 125 amino acids of the 590
amino acid polypeptide are hydrophobic, whereas the rest
of the protein is hydrophilic. The protein is negatively
charged (–0.758) at pH 7, with an average isoelectric
point of 6.9. At the C-terminus, two Zn finger-like domains
can be seen. One consists of a Cys/Cys–Cys/Cys and the
other of a Cys/Cys–His/Cys sequence. This motif has also
been found in several other eukaryotic proteins (Figure
2). In addition, three CDC28/CDC2-like phosphorylation
sites [SPPR (CDC28 site of S.cerevisiae), PDSP and
PTTP (CDC2 sites of Schizosaccharomyces pombe)] were
identified in the middle region of the polypeptide (Figure
2). A search for SamB sequence similarity in the databases
detected a hypothetical protein (YMR100w) in S.cere-
visiae.Thetwofungalproteinssharedsequencesimilarities
at the N- and the C-termini, with 70% similarity (49%
identity) and 75% similarity (62% identity), respectively.
In addition to the conservation of the primary sequences
of the genes, the secondary structure and hydrophilicity
predictions, the CDC28 sites and the Zn finger-like domain
were also similar in both proteins (Figure 2). These
findings suggest conserved functions in A.nidulans and in
S.cerevisiae.
The SamB protein resides in the cytoplasm
The analysis of the SamB protein revealed a hydrophobic
region at the N-terminus and a Zn finger-like domain at
the C-terminus. These domains suggested an association
of the protein with a membrane or with the nucleus in the
case the Zn finger domain functions in DNA binding. To
test this, we analyzed the SamB protein in A.nidulans and
localized the protein by secondary immunofluorescence
in the cell (Figure 3). We introduced the hemagglutinin
(HA) epitope of the human influenza virus into the
BamHI restriction site of samB (pMir25). This construct
complemented the samB defect in transformation experi-
ments, indicating that the 33 additional amino acids
inserted into SamB did not interfere with the biological
function of the protein. A transformant with a single
integration of the construct (SMI52-7) was used to detect
the SamB protein in vitro. Protein extracts were prepared
and separated on a polyacrylamide gel. Using monoclonal
anti-HA antibodies in a subsequent Western blot analysis,

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M.Kru ¨ger and R.Fischer
Fig. 1. Molecular analysis of samB. (A) Partial restriction map of the samB locus. The location of the transcript and the open reading frame are
indicated. (B) Detection of the samB transcript in RNA isolated from hyphae of the A.nidulans wild-type (samB?) strain FGSC26. Twenty-five µg of
total RNA were separated on a 1% agarose gel (glyoxal), transferred to a nylon membrane and processed for transcript detection. As a probe, we
used the32P-labeled EcoRV fragment of samB. (C) Sequence of the samB locus. The 4.1 kb EcoRI fragment was sequenced on both strands and
compared with the cDNA sequence. The start sites of three cDNAs are marked above the sequence (*). Putative TATAAA-like and CAAT boxes are
indicated (underlined). The poly(A) addition sites (*) were deduced from the cDNA sequences. The amino acid sequence of the open reading frame
is indicated below the DNA sequence in the one-letter code. The point mutation leading to a stop codon detected in SMI20 is boxed. The sequence
data reported here have been submitted to the DDBJ/EMBL/GenBank databases under accession No. AJ000996.
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Morphogenesis in fungi
Fig. 2. Properties of the SamB protein. (A) Hydrophilicity prediction after Kyte–Doolittle. (B) Comparison of samB and the yeast homolog. Regions
of high sequence similarity, the hydrophobic amino-terminus, the Zn finger-like domain and putative CDC28 sites are indicated. (C) SamB shares a
Zn finger-like domain with the yeast protein Mub1 (accession No. gp Z49807), the Drosophila proteins DEAF-1 (Gross and McGinnis, 1996) and
Nervy (Feinstein et al., 1995), the human MTG8 protein (Miyoshi et al., 1993) and the mouse RP-8 protein (accession No. gp U10903). The
alignment was achieved using the Jothun Hein method (Meg Align program, Laser Gene Navigator, DNA STAR Inc., Madison, WI). The conserved
cysteine and histidine residues are denoted in bold letters below the sequences.
we detected a specific band in SMI52-7 which expressed
the SamB::HA fusion protein, as compared with FGSC26
which only expressed the non-tagged SamB protein. The
calculated apparent Mrwas 80 kDa, which is slightly
higher than the predicted Mrof 70 kDa (66 kDa plus
the HA epitope). The protein was not very abundant.
Expression was also studied in an apsA deletion strain
which was transformed with the SamB::3HA construct.
Several independent transformants were analyzed on a
Western blot. No significant difference could be detected
in comparison with the expression in an apsA?strain.
The A.nidulans strains expressing SamB::3HA were also
processed for immunostaining with monoclonal anti-HA
antibodies and Cy3-labeled anti-mouse secondary anti-
bodies for detection. Granular staining of the cytoplasm
was observed (result not shown). A stronger signal was
obtainedwithastrain(SMI52-10)withseveralintegrations
of the construct. An association with known subcellular
structures such as microtubules or actin was not obvious
(Figure 3). The protein was not clearly excluded from
nuclei.
SamB is involved in fungal morphogenesis
The A.nidulans SamB protein showed significant homo-
logy to the hypothetical yeast protein YMR100w. To
compare the phenotype of the samB mutant with the
phenotype of a loss-of-function mutant of S.cerevisiae,
we deleted the YMR100w coding region in the haploid
207
yeast strain MH272-1da. We amplified the S.cerevisiae
HIS3 marker gene using HIS-specific oligonucleotides
with 50 bp overhangs at the 5? end derived from the
flanking sequences of the coding region of YMR100w.
The resulting PCR fragment was gel-purified and used for
yeast transformation. In one of 13 transformants, the
predicted gene replacement led to the deletion of the
coding region (result not shown). The haploid transformant
wasviable.ThenameMUB1(multi-budding)wasassigned
to the gene for the observed phenotype. The morphology
of ∆MUB1 mutant cells was studied in detail and compared
with the A.nidulans samB phenotype.
Comparison of the S.cerevisiae and A.nidulans
phenotypes
The yeast deletion strain MK1α-H12 and the yeast wild-
type strain MH272-1dα were grown in liquid culture at
30 and 37°C, and the budding pattern was analyzed in
late exponential growth. We found that normal axial
budding occurred in both haploid strains. In addition,
bipolar and multi-budded cells were observed in which
new buds were generated before cytokinesis was com-
pleted. The multi-budded cell clusters even remained
when the cells were sonicated. The relative abundance of
abnormal budded cells was increased in the MK1α-H12
deletion strain in comparison with the wild-type. The
effect was more pronounced at 37°C (Figure 4, Table I).
A comparable phenotype was also observed in A.nidulans

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M.Kru ¨ger and R.Fischer
Fig. 3. Immunological detection of the SamB protein in A.nidulans.
(A) Introduction of the HA epitope into SamB. (B) Detection of
SamB::HA (*) in crude protein extracts of the transformed strain
SMI52-7. Germinated conidia were harvested and protein extracts
prepared. Twenty-five µg of total protein were separated on a 10%
SDS–PAGE and blotted onto a nitrocellulose membrane. The
HA-tagged SamB protein was visualized using monoclonal anti-HA
antibodies, affinity-purified anti-mouse secondary antibodies and a
chemiluminescent detection system. Lane 1, FGSC26; lane 2,
SamB::HA expressed in the apsA?strain SMI52-7; lane 3, SamB::HA
expressed in the apsA mutant strain SMI49-3. (C) Localization of
SamB in vivo. Conidia of FGSC26 (left panel, a and b) and SMI52-10
(right panel, c and d) were germinated on cover slips and processed
for secondary immunofluorescence. Monoclonal anti-HA antibodies
and Cy3-labeled, affinity-purified secondary anti-mouse antibodies
were applied. (a and c) phase contrast, (b and d) samB fluorescence of
the same germlings. Bar ? 5 µm.
carryingthesamBmutation.ThesamBmutantwasisolated
originally as a suppressor of the apsA mutation which led
to an increase of spore production of the oligosporogenous
apsA strains (Kru ¨ger and Fischer, 1996). The phenotype
of the samB mutation in an apsA?strain with respect to
asexual spore production or conidiophore development
differed only slightly in comparison with a wild-type
strain. This could have been due to only a partial loss of
function of SamB after mutagenesis. To investigate the
defect of the protein, we PCR-amplified the samB mutant
allele and compared the sequence with the genomic DNA
sequence of a wild-type strain. Twenty independent PCR
clones of the mutant were pooled and used as templates
208
Fig. 4. Phenotypes of a S.cerevisiae wild-type strain (A) and the yeast
deletion strain MK1α-H12 (B). (A and B) Low magnification view of
two cultures in the late exponential growth phase (bar ? 15 µm).
Strains were grown at 37°C. Multi-budded cell clusters are indicated
by arrows. (C–H) Yeast cells displaying different budding patterns
(bar ? 5 µm). The relative abundance of the different types in a
wild-type culture and in the knock-out mutant culture are denoted in
Table I.
Table I. Comparison of the budding pattern of a yeast wild-type strain
and the MUB1 deletion strain (MK1α-H12)
Strain/growth
temperature
Regular budding Regular bud
0 or 1 bud
Multi-budded and/or
irregular bud sitessite 2 buds
Wild-type/30°C
Knock-out/30°C 80.8
Wild-type/37°C
Knock-out/37°C 55.9
92.9 5.9
14.3
18.7
28.5
1.2
4.9
3.2
15.6
78.1
The yeast strains were grown in liquid YPD medium at 30 and 37°C
and analyzed in the late exponential growth phase. In each case, ?250
cells were counted. A multi-budded complex was counted as one.
Numbers are given in percent. For a description of the phenotypes see
Figure 4.
for the sequencing reaction. This should allow for valid
sequencing not disturbed by errors introduced during the
amplification. It was found that in the mutant a point
mutation at base pair 2519 changed the codon TGG
(tryptophan) to the stop codon TAG. The observed trans-
ition from G to A is consistent with the method used (di-
ethylsulfate as mutagen) (Kru ¨ger and Fischer, 1996). This
transition results in the expression of a truncated protein
in which the carboxy-terminus harboring the Zn finger-
like domain is missing (expression of the protein has not
been examined). The truncation should severely interfere
with properties of the protein and can be considered non-
functional. The multi-budding phenotype of the yeast
mutant prompted us to re-investigate the phenotype of the
samB mutant using germinating spores. In wild-type cells,
spores produce a germ tube which is populated by nuclei
through the coordinated action of nuclear division and
nuclear migration (Suelmann et al., 1997). After the third
round of mitosis, a septum is formed at the rear of the
germ tube, separating the spore from the growing cell.
The remaining spore usually produces a second germ tube
opposite the first protrusion. In a wild-type population of
germlings at the eight nuclei stage, ?70% of the cells