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J Biol Chem, Vol. 275, Issue 1, 167-176, January 7, 2000
From the Department of Biology, University of California,
Santa Cruz, California 95064
By using the process of Repeat-induced Point
mutation (Selker, E. U., and Garrett, P. W. (1988)
Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874), we
inactivated vma-1, the gene encoding subunit A of the
V-ATPase of Neurospora crassa. Two vma-1 mutant strains were characterized. One was mutated at multiple sites, did not
make a protein product, and produced spores that only rarely
germinated. The other had four point mutations, made a protein product,
and produced viable spores. Neither strain had detectable V-ATPase
activity. The vma-1 mutant strains did not grow in medium
buffered to pH 7.0 or above or in medium supplemented with the cation
Zn2+. They were completely resistant to inhibition by
concanamycin C, supporting our hypothesis that the V-ATPase is the
in vivo target of this antibiotic. Inactivation of the
vma-1 gene had a pronounced effect on morphology and
development of the organism. In the mutants tip growth was inhibited,
and multiple branching was induced. The vma-1 mutant
strains could not differentiate conidia or perithecia. They could grow
slowly as mycelia and could donate nuclei in a sexual cross. A mutation
in the plasma membrane ATPase, which suppressed the sensitivity of wild
type N. crassa to concanamycin, also proved effective in
suppressing the sensitivity of a vma-1 null mutant to basic
pH but did not correct the morphological defects.
Vacuolar H+-ATPases
(V-ATPases)1 are large,
complex enzymes found on multiple members of the endomembrane system of
eukaryotic cells and on the plasma membranes of many specialized cells
(1, 2). They hydrolyze ATP and pump protons across membranes to acidify
cellular compartments. They have been implicated in a number of
cellular processes, including protein sorting, receptor-mediated endocytosis, zymogen activation, and pH and calcium homeostasis. Studies of V-ATPases in fungi have primarily focused on the enzyme in
vacuolar membranes. These organelles are important as storage reservoirs for basic amino acids and polyphosphate, as sites for protein degradation and turnover, and as sequestration sites of potentially toxic ions, especially Ca2+ (3-5).
Because of the central role of the V-ATPase in cellular metabolism, we
anticipated that genes encoding it would be essential, and in animal
cells that appears to be the case. In Drosophila melanogaster, inactivation of vha55, the gene encoding
subunit B of the V-ATPase, resulted in a larval lethal phenotype (6). In Dictyostelium discoideum, a slime mold, the failure to
obtain a V-ATPase knockout strain combined with characterization of
strains carrying antisense RNA for the proteolipid c subunit of the
V-ATPase led to the conclusion that V-ATPase genes were probably
essential in this organism (7). Similarly, we reported that
vma-1, the gene encoding the catalytic subunit A of the
V-ATPase, was indispensable in the filamentous fungus Neurospora
crassa. In that study we generated a RIP/Rescue strain, which
contained an inactivated vma-1 gene at the endogenous site
and a functional second copy at an ectopic site. When the RIP/Rescue
strain was crossed with the wild type, the mutated vma-1
gene behaved like a simple recessive lethal (8).
However, V-ATPase genes are clearly not essential in the yeast,
Saccharomyces cerevisiae, where deletion strains have been characterized in considerable detail. Perhaps the most defining and
useful phenotype of VMA deletion strains in yeast is the
response to pH; VMA delete strains grow reasonably well in
acidic medium (pH 5.5) but fail to grow in basic medium (above pH 7)
(9, 10). The strains are sensitive to high concentrations of
Ca2+ (11) in the medium or of heavy metals such as
Zn2+, Mn2+, and Cu2+ (12-14).
Although not strictly pet More recently the VMA1 gene was disrupted in Ashbya
gossypii, a filamentous fungus related to S. cerevisiae. Surprisingly, in contrast to S. cerevisiae,
the deletion strain of A. gossypii did not exhibit a
pH-conditional lethal phenotype nor was it particularly sensitive to
elevated Ca2+ levels in the medium. However, the mutant
strain grew more slowly and more compactly than wild type, secreted
amino acids typically found in the vacuole, and failed to form
generative spores (17).
By serendipity, we have now obtained viable strains of N. crassa, in which the vma-1 gene has been inactivated.
Our previous failure to obtain such strains can be explained by the low
germination efficiency of ascospores containing the mutated gene (8).
The biology of N. crassa is significantly different from
S. cerevisiae, the organism in which most studies of
vma null strains have been done. N. crassa is an
obligate aerobe. We wanted to determine if loss of the vacuolar ATPase
caused the metabolic phenotypes observed in yeast VMA
mutants, e.g. failure to grow on high concentrations of a
non-fermentable carbon source. The life cycle of N. crassa involves significant cellular differentiation and morphological complexity. In this report vma null mutant strains have been
examined to see if the vacuolar ATPase has a role in the development of the organism. In addition the availability of the mutant strains allowed us to see if the vacuolar ATPase is the primary target, in vivo, of the widely used antibiotic concanamycin (18). We have conducted experiments to determine if this antibiotic affects strains that lack the vacuolar ATPase and if the phenotype of the
vma mutant strains is similar to the phenotype of wild type strains growing on concanamycin.
N. crassa Strains--
The properties of the strains used in
this work are summarized in Table I. Wild
type and marker strains came from the Fungal Genetics Stock Center
(FGSC, University of Kansas Medical Center, Kansas City, KS). Strain
vma-1RR1 was described in Ferea et al. (8) and ccr4-5 in
Bowman et al. (19). Strain RR24-7-48a was generated from a
cross between vma-1RR1a and a strain with genetic markers for
his-1, al-3, and inl, linked genes on
linkage group V near vma-1, given to us by Dr. Robert
Metzenberg (Stanford University). Bump was constructed in collaboration
with Dr. Metzenberg in an earlier attempt to inactivate
vma-1 by sheltered RIP (20). We inserted a 2.4-kilobase pair
SstI fragment of genomic DNA from vma-1 (21) into
the plasmid pCSN43 (22), which has the hph gene that encodes
hygromycin phosphotransferase and confers resistance to hygromycin B in
N. crassa. The vma-1 fragment terminated at amino
acid residue 484 (of 607) and presumably did not make a functional gene
product. This plasmid, pRIP1, was introduced into the sheltered RIP
host. Crossing of the transformant to wild type 74A resulted in
recovery of the strain bump, which exhibited an extreme colonial growth
habit.2 Genetic analysis
showed that bump contained a wild type vma-1 gene at the
endogenous locus on linkage group V and that the plasmid carrying the
hph gene together with the partial vma-1 gene had integrated very near arg12 on linkage group II. Our
hypothesis is that pRIP1 interrupted a gene of unknown function,
causing the bump phenotype. Thus, fortuitously, bump had two copies of vma-1, a functional copy at the endogenous location and a
non-functional copy at an exogenous location, making it a suitable
strain for inactivation of the vma-1 gene by RIP as
described under "Results." The origin of pvn (putative
vma null) strains is described under "Results."
Genetic Manipulations, Scoring of Phenotypes, and Growth
Tests--
For standard genetic analyses, i.e. crossing and
analyzing progeny, the procedures of Davis and de Serres (23) were
used. Because bump and pvn strains grow slowly and do not conidiate, they were maintained as heterokaryons with the sterile am1
helper strain (FGSC 4564). The heterokaryons grew like wild type, but
only the nuclei without the am1 mutation could
participate in a sexual cross (24). Typically, spores from a sexual
cross were spread on agar plates, heated for 35 min at 60 °C to
induce germination, and picked to 1 ml of liquid medium. After 5 days
of growth at 30 °C and 3-7 days more at room temperature,
phenotypes were tested. Progeny that grew well, i.e. not pvn
strains, were spot tested on agar plates containing Vogel's medium N
(VM, a minimal medium salt solution) supplemented with 2% sorbose to
induce colonial growth, 0.05% fructose, 0.05% glucose, and 0.025%
inositol and other supplements as needed. Slow-growing pvn strains were
usually tested in 1 ml of liquid medium (VM with 2% sucrose plus
supplements) or less frequently on agar plates with VM and 2% sucrose
as the carbon source. Am
For quantitative comparisons of the growth rates of wild type and pvn
strains, we measured colony diameters on agar plates containing VM and
2% sucrose with supplements as indicated under "Results." Plates
were inoculated with the wild type from a conidial suspension, and
plates were inoculated with the pvn strain from a plug of culture from
a colony on an agar plate. Colony diameter was measured for an 8-h
interval following the lag phase for the wild type and at 24-h
intervals for the pvn strains during growth at 30 °C. To measure
growth as the increase in mass, cells were grown in aerated liquid
medium. Wild type cultures were inoculated with 106 conidia
per ml. Because pvn strains have an unusual growth morphology and do
not produce conidia, an alternative procedure was necessary. Bits of
mycelium were scraped off an agar surface or tweezed apart from a
liquid tube culture and grown at 25 °C for 2-4 days in 25 ml of
medium on a reciprocal shaker. The resultant balls of mycelium were
suspended in fresh medium and macerated for 30 s in a Waring
blender, yielding a uniform suspension of mycelia. An aliquot of this
mycelial suspension was collected and extracted with acetone to
determine the dry weight. Liquid cultures were inoculated with the
suspension to give 0.025 mg dry wt of mycelium/ml of medium. To measure
doubling times cells were grown at 25 °C in 4 liters of medium with
forced air. Dry weights were determined on 50-100-ml aliquots taken
during the exponential growth phase, between 0.1 and 1.2 mg/ml. In some
experiments growth yield was compared as the dry weight of mycelium
produced in 25 ml of liquid medium grown 2-4 days at 25 °C on a shaker.
Analysis of vma-1 in pvn Strains--
To demonstrate the
presence of a single, mutated copy of vma-1 in pvn strains,
we used Southern blotting and DNA sequencing. Genomic DNA was extracted
from N. crassa by the method of Oakley et al.
(25), digested with BamHI, electrophoresed, and transferred to Nytran using a TE-80 Transvac vacuum blotter (Hoeffer Scientific, San Francisco). The blot was probed with a radioactive 1632-nucleotide BglII/BamHI fragment of vma-1 (21),
using the method of Feinberg and Vogelstein (26). The endogenous copy
of vma-1 was amplified by polymerase chain reaction with
specific primers, 5'-CAATACCTCTACCACCACC-3' and
5'-CAGACAGCCCATGCATCATGC-3', and subcloned to pCR2.1 from the Original
TA Cloning Kit (Invitrogen, Carlsbad, CA). The cloned DNA was sequenced
on a Perkin-Elmer ABI310 Sequencer, using the Big Dye dye terminator
kit (Perkin-Elmer).
Arginine Content in Cells and ATPase Activity in
Membranes--
The arginine content in whole cell extracts was assayed
as described (19). Vacuolar membranes, plasma membranes, and
mitochondria were isolated as described (27) and modified (19). In
addition, the supernatant remaining after pelleting plasma membranes at 49,000 × g was subjected to a final high speed
centrifugation step (1 h at 40,000 rpm in a Beckman Ti60 rotor,
161,000 × gmax) to collect a "light
membrane" fraction, expected to contain any membranes not previously
pelleted and to be enriched in endoplasmic reticulum. Protein and
ATPase activities were assayed as before (27).
Microscopy--
To visualize the growing front of mycelium,
cultures were grown as colonies on agar plates. Regions of hyphae were
photographed directly by bright field microscopy. To examine vacuoles,
mycelia were grown overnight in liquid and observed under Nomarski
optics. We used Kodak TMY 400 print film and a Leitz Aristoplan
microscope and camera.
Isolation of a vma-1 Null Strain from the RIP/Rescue
Strain--
To generate a strain of N. crassa that lacked a
functional V-ATPase, we used the process of RIP (28). N. crassa is a haploid organism, which readily takes up DNA by
transformation and incorporates it into the genome either by homologous
recombination or more frequently by recombination at random locations.
When a second copy of an endogenous gene is introduced by
transformation and the transformant is crossed to another strain, a
unique process called RIP (Repeat-induced
point mutation) scans the genome and introduces multiple
point mutations into both copies of the duplicated regions of DNA.
Because the efficiency of RIP varies, individual progeny from such a
cross can have many, few, or no mutations in the duplicated gene. By
using this procedure, we earlier generated a RIP/Rescue strain,
vma-1RR1, in which the endogenous copy of vma-1 was
inactivated by multiple point mutations and a second, functional copy
was introduced on a different chromosome. The strain grew like wild
type. Attempts to generate a strain that contained only the inactivated
endogenous vma-1 gene by genetics were unsuccessful, leading
us to propose that vma-1 was an essential gene in N. crassa (8).
A more recent cross gave a different result. We exploited the fact that
the vma-1 gene is tightly linked (less than 1%
recombination) to the am gene on chromosome V. The
RIP/Rescue strain, vma-1RR1, is am
To determine whether the slow growth phenotype was caused by
inactivation of the vma-1 gene, we used both genetic and
biochemical tests. First, strain pvn1-121A was crossed to the wild type
74a to look for linkage of slow growth to Am
In a second test we found that a functional ectopic copy of
vma-1 rescued the slow growth phenotype in pvn1. In a
genetic cross we replaced the chromosome V that carried the
vma-1RIP gene in strain vma-1RR1 with a chromosome V
carrying wild type genes for am and vma-1 and
mutant marker genes for his-1, al-3, and
inl loci. This new strain, RR24-7-48a, was crossed to
pvn1-121A (Table III). All the progeny
grew like wild type. Among the Am+ progeny, which were
predicted to be vma-1+, half were
HygS and half were HygR, indicating that they
were able to grow with or without the ectopic vma-1 gene.
The Am
Our interpretations of the genetic experiments were supported by
Southern blot analysis and sequencing. Genomic DNA digested with
BamHI and probed with vma-1 DNA revealed a single
band in the wild type strain and two bands, corresponding to the
endogenous and the ectopic genes, in the RIP/Rescue parent (Fig.
1A). pvn1-121A had a single
band, of the size predicted for the endogenous gene on chromosome V. Finally we used the polymerase chain reaction to clone the endogenous
vma-1 gene from strain pvn1-121A. The sequence was identical
to that previously reported for the vma-1RIP gene in
strain vma-1RR1 (Fig. 1C and Ref. 8). The gene contained 42 point mutations, one of which converted amino acid residue 130 (of 607)
into a stop codon.
We concluded that pvn1-121A was indeed a viable vma-1 null
strain of N. crassa. The pvn phenotype was tightly linked to
am Isolation of a vma-1 Null Strain from the Mutant Strain
Bump--
The strain bump is a tight colonial mutant that was
generated by an alternative approach to inactivate vma-1 as
described under "Experimental Procedures." In essence, bump
contains a normal endogenous vma-1 gene on chromosome V and
a partial vma-1 gene, incorporated via a plasmid with an
hph gene (confers resistance to hygromycin), on linkage
group II near the arg12 locus. Thus, bump was a good
candidate to use in RIPing experiments, and it fortuitously led us to a
second pvn strain, pvn2, which differs in certain properties from pvn1.
In an effort to map the chromosomal location of bump more precisely, we
crossed it to an arg12
Southern blot analysis confirmed that a single copy of vma-1
DNA was present at the endogenous locus in strain pvn2 (Fig. 1B), and sequence analysis confirmed that this DNA was
mutated (Fig. 1D). RIPing in strain pvn2 was light. Four
nucleotides within the coding region of the gene were mutated from G to
A, predicting amino acid residue changes in subunit A of G170S, D266N,
G375S, and G392S. No stop codons were introduced, suggesting that pvn2, unlike pvn1, could make a protein product. Whether the protein would be
functional was harder to predict. All four of the affected residues are
conserved among eukaryotic V-ATPases but not in the comparable residue
of the homologous
We concluded that the slow growth phenotype of pvn2 was due to mutation
of the vma-1 gene by RIP. In contrast to pvn1, the pvn2
spores germinated efficiently, and a protein product was likely to be
made. Because the strain grows so poorly, it probably produces either
no active V-ATPase or enzyme with minimal activity.
Growth Properties of pvn1 and pvn2--
To examine the effect of
gene inactivation on the growth of N. crassa we surveyed a
number of conditions found to be inhibitory to VMA deletion
strains in S. cerevisiae. In nature filamentous fungi grow
primarily by apical extension of hyphae over a solid surface. For most
of our growth experiments we measured the increase in colony diameter
on an agar plate. The pvn strains grew slowly compared with wild type,
but the visible growth on plates was considerably more vigorous than in
standing liquid medium. In some experiments, growth was measured as the
increase in mass in an aerated liquid culture.
The two pvn strains behaved like true pH conditional lethal mutants
(Fig. 2A), a hallmark of the
yeast VMA deletion mutants (9, 10). On agar medium adjusted
to pH 7.2, wild type grew somewhat more slowly than at pH 5.8, and the
two pvn strains did not grow at all. Measurement of growth as the
increase in mass in a shaking liquid culture yielded qualitatively
similar results (Fig. 2B). The wild type grew equally well
over a wide pH range, from 4 to 7; the two pvn strains grew best at pH
5.5, falling off sharply both above and below this value. We have shown
that concanamycin A, a specific inhibitor of V-ATPases, confers a pH conditional phenotype on growth of wild type N. crassa (19). Concanamycin slows growth of 74A at pH 5.8 (standard minimal medium) and prevents it at pH 7.2. If the V-ATPase is the only target of
concanamycin in the cell, we would predict that a vma null strain should be completely resistant to the antibiotic. Pleasingly, both pvn1 and pvn2 strains grew identically with or without
concanamycin in the medium at pH 5.8 (Fig. 2A).
The pvn strains were initially isolated and characterized on the basis
of their unusually slow growth in standing liquid medium, where the
mycelia failed to reach the surface of 1 ml of liquid in 2 weeks.
Growth rates on agar plates (pH 5.8) at 30 °C were also strongly
affected in the pvn mutants. Rates of increase in colony diameter were
4.2 mm/h for wild type, 0.5 mm/h for pvn1-121A, and 0.63 mm/h for
pvn2-53-19A (Table V). The effects on
growth as an increase in mass of cells were less severe. The doubling times for the pvn mutants were no more than twice that for the wild
type in either minimal or complete medium (Table V), similar to the
results reported for VMA disruptants of S. cerevisiae and A. gossypii (9, 17).
The sensitivity of the pvn strains from N. crassa to several
other compounds is summarized in Table
VI. Growth was measured as the percent increase in colony diameter,
compared with the colony size on minimal medium as the 100% control.
Inositol, reported to stimulate growth of a VMA1 deletion
strain in S. cerevisiae (11), had no effect on the N. crassa pvn strains. 0.1 M NaCl, expected to inhibit,
also had no effect. However, a high concentration of NaCl (1.0 M) inhibited growth of wild type significantly (68%) and
the vma-1 mutants almost completely. A number of divalent cations are sequestered in fungal vacuoles, thus maintaining low, non-toxic cytosolic concentrations of these substances. Several ions
have been reported to be potent inhibitors of growth in yeast VMA deletion strains (12-14). Only 4 mM
Zn2+ was particularly potent against the N. crassa pvn strains. Mn2+ (3 mM) and
Fe2+ (3 mM) were weakly inhibitory, and even
high Ca2+ (100 mM) inhibited the pvn strains
only 50% while also mildly affecting the wild type. Cu2+
appeared to stimulate growth of the vma-1 mutants. Compared
with the wild type, the N. crassa pvn strains grew
reasonably well on non-fermentable carbon sources, 1.5% glycerol and
50 mM sodium acetate, as measured by linear extension.
However, the mycelial density was considerably reduced in both wild
type and pvn mutants on these media. When grown on a highly enriched,
undefined medium (complete), all three strains grew marginally faster
than the 100% controls on minimal medium, and the mycelial mats looked more filled in on the rich medium.
Lack of V-ATPase Function in pvn1 and pvn2--
Arginine, together
with other basic amino acids, is stored at high concentrations in
fungal vacuoles. Accumulation of arginine is dependent on the proton
gradient generated by the V-ATPase (29). Consequently, a convenient and
straightforward method to assess in vivo function of the
V-ATPase is to measure the acid-soluble arginine in whole cell
extracts. Strains pvn1 and pvn2 accumulated 5.7 and 5.2 nmol of Arg/10
mg dry weight of cells, respectively, approximately 1/5th the amount
found in 74A (28.7 nmol/10 mg) or the RIP/Rescue strain vma-1RR1 (26.7 nmol/10 mg) and similar to the amount found in cells grown in
concanamycin A to inhibit the V-ATPase (19). These results indicated
lack of a functional V-ATPase in either pvn1 or pvn2. By using our
standard procedures (27), we isolated membrane fractions containing
gradient-purified mitochondria, vacuolar membranes, plasma membranes,
and "light" membranes (endoplasmic reticulum and others), and
compared ATPase activities in strains 74A and pvn2-53-19A. We thought
it necessary to collect all the membrane fractions because our
procedure for isolation of vacuolar membranes depends on having
osmotically active vacuoles that become heavy when cells are lysed in 1 M sorbitol and pellet with the dense mitochondria. In the
absence of V-ATPase activity the vacuoles do not accumulate the same
amount of solutes (e.g. arginine and other basic amino
acids) and probably do not behave as dense organelles in the
fractionation procedure. Instead, isolated vacuolar membranes, which
have a low density, would be expected to be in the light membrane
fraction. In 74A the V-ATPase specific activity (pH 7.5, CCC-sensitive)
was 2.0 µmol/min/mg of protein in vacuolar membranes and less than
0.1 in the other membrane fractions. Strain pvn2-53-19A had no
detectable V-ATPase activity in any membrane fraction, as expected if
the strain produces no active V-ATPase.
pvn Mutants Demonstrate Striking Differences in Morphology and
Development--
In N. crassa, the effects of disrupting
the vma-1 gene on morphology and development were dramatic.
Both pvn strains had a distinctive colony morphology on 2% agar (Fig.
2A). Although the wild type rapidly spreads across the
surface, primarily by hyphal tip extension, and produces aerial hyphae
that result in a cottony appearance, the pvn strains progressed slowly
and produced few aerial hyphae. Then the colony filled in behind the
growing front, resulting in a characteristic layered appearance.
Comparison of the growth fronts at the microscopic level showed a clear
difference in branching patterns (Fig.
3). Wild type grows primarily by apical extension and produces side branches at infrequent intervals, resulting
in a rapid advance across the agar surface. In the pvn strains
branching was primarily dichotomous and frequent, resulting in a
crowded growth front that advanced forward slowly.
Vacuoles in VMA disruptants of S. cerevisiae (10)
and A. gossypii (17) were reported to appear normal. This
was not the case in the pvn strains of N. crassa. Compared
with wild type vacuoles those in the two pvn strains were more
irregular, often misshapen, and frequently appeared to be multilamellar
(Fig. 4). Together, the cell
fractionation results and microscopic analysis of vacuoles strongly
suggested that vacuolar morphology was significantly altered by
inactivation of vma-1 in N. crassa.
The pvn strains had also lost the capacity for some types of cellular
differentiation. N. crassa has an interesting life cycle that requires the development of several different morphological structures (30). When nutrients are available, wild type N. crassa grows vegetatively as filamentous hyphae that fuse and intertwine to form a mycelial mat, the defining state of a filamentous fungus. Environmental conditions such as nutrient depletion or desiccation induce a conidiation pathway. Aerial hyphae form, followed
by differentiation into asexual spores called conidia. Conidia can
germinate and grow into a new mycelium or function as a male parent in
a sexual cross. On crossing medium, which is low in nitrogen, N. crassa enters a sexual cycle. Hyphae of opposite mating types (A
and a) fuse and form a complex fruiting body, the perithecium. Within
this structure the nuclei fuse to form the only diploid cell in the
life cycle and immediately undergo meiosis. The four meiotic products
divide, yielding eight nuclei, each of which develops into a large
tough spore called an ascospore. The ascospores can lie dormant for
years or be induced by heat shock to germinate and go through the
vegetative life cycle.
pvn1 and pvn2 mutants were blocked at several stages of development in
the life cycle. They produced only short aerial hyphae and never formed
conidia, the asexual spores. They could not differentiate into a
fruiting body and thus could not function as a female parent. However,
they did function as a male parent, donating a nucleus in a sexual
cross to a fertile strain such as 74A. In such a cross mature
ascospores were produced. The wild type spore progeny germinated efficiently. Consistent with the results described above, pvn1 and pvn2
mutant spores differed significantly in germination rate. Spores
carrying the pvn1 mutation germinated at low frequency (0.5-1.0%),
but spores with the pvn2 mutation germinated as efficiently as wild
type. Upon germination, both strains grew slowly, forming highly
branched hyphae as described above.
By using the am1 helper strain, which carries a
mating type mutation (24), we were able to investigate the consequences
of crossing pvn mutants to themselves. We constructed heterokaryons containing nuclei from the am1 helper strain and
from each of the pvn strains. These heterokaryons grew like wild type
and made abundant conidia and protoperithecia, indicating that the
vma-1RIP alleles were recessive. By using these
heterokaryons, we made pvn1-121A × pvn1-121a and pvn2-53-19A × pvn2-53-19a crosses. Although the helper strain allows for formation
of perithecia, it does not contribute to development of asci and
ascospores. In both pvn crosses ascus development was arrested in
meiotic prophase.4 In
pvn1-121A × pvn1-121a the homologous chromosomes showed little or
no pairing. In pvn2-53-19A × pvn2-53-19a arrest was somewhat later. Chromosomes did pair, and some anaphase stages and chromosome segregation were observed. However, in both crosses asci aborted prior
to development of individual ascospores, and no mature ascospores were formed.
In summary, inactivation of vma-1 resulted in multiple
defects in morphogenesis of N. crassa. Pvn strains grew
slowly, showed altered hyphal branching patterns, failed to
differentiate conidia or perithecia, arrested in meiosis, and in the
case of pvn1 spores germinated only rarely. These results point to an
important role of the V-ATPase, either direct or indirect, in all of
these processes.
Partial Suppression of the pvn Phenotype by a pma-1 Mutation and by
Sorbitol--
To begin to dissect the role of the V-ATPase in these
processes, we sought conditions that could suppress the pvn phenotype. By having previously isolated and characterized mutants that could partially suppress sensitivity to concanamycin A (or C) (19), we first
tested these mutants for the capacity to suppress the pvn phenotype.
The ccr strains are mutated in pma-1, the gene encoding the
plasma membrane H+-ATPase. They allow N. crassa
to grow in the presence of concanamycin at basic pH. The effect of the
mutation is to change the kinetic behavior of the plasma membrane
H+-ATPase. In the ccr mutant strain the
Km for ATP is lower, and the curve that describes
rate of hydrolysis versus ATP concentration is hyperbolic,
as opposed to the sigmoid curve observed in the wild type. We have
suggested that the mutants are resistant to concanamycin because the
altered plasma membrane H+-ATPase can use low levels of ATP
more efficiently than the wild type enzyme. Thus, substances like
calcium or divalent cations, released from the vacuole into the cytosol
when the V-ATPase is inhibited by concanamycin, can be pumped out of
the cell and prevented from attaining toxic concentrations in the cytosol.
We constructed double mutants of pvn2-53-19 and ccr4-5 by a genetic
cross. (We were unable to obtain pvn1, ccr4-5 double mutants, presumably due to the deficiency in spore germination.) The most significant effect of the ccr mutation was suppression of the pH-sensitive phenotype. In the experiment shown in Fig.
5, pvn2-53 did not grow at pH 7.0 and
above, but the double mutant grew over the same range of pH as the wild
type. Growth of pvn2-53 and of pvn2-53, ccr4-5 was also compared under
the conditions given in Table V. In these tests the double mutant grew
marginally better, 10-20%, than the pvn2 mutant (data not shown) and
was completely resistant to concanamycin. The double mutant had the
same morphological and developmental deficiencies as the pvn2 single
mutant strain. Thus, the ccr mutation was effective in suppressing the
sensitivity of pvn to basic pH yet had little effect on other
phenotypes caused by inactivation of vma-1.
While testing growth of pvn strains on different media, we added 1 M sorbitol to the medium as a test of osmotic sensitivity. After several days of growth conidia were produced by the pvn2 strain
(or pvn2, ccr4-5 double mutant) but not the pvn1 strain (Fig.
6). Thus, some signal brought about by
the high concentration of sorbitol was able to overcome the deficiency
of the pvn2 strain in forming conidia. We are investigating the
explanation for this surprising phenomenon.
Phenotype of vma-1 Null Mutants in N. crassa--
To our
knowledge, deletion strains for genes encoding V-ATPase subunits have
been described in only three organisms. Deletion of the gene encoding
subunit B in D. melanogaster resulted in an embryonic lethal
phenotype (6). In the yeast S. cerevisiae and the closely
related filamentous fungus A. gossypii, deletion of V-ATPase
genes was not lethal but did cause a number of growth defects. We have
previously reported that vma-1, which encodes subunit A of
the vacuolar ATPase, appeared to be essential in the filamentous fungus
N. crassa (8). However, in the present paper we have
succeeded in inactivating vma-1, and we show that the gene
is not essential for vegetative growth but is necessary for normal
morphological development.
Inactivation of the V-ATPase had pleiotropic effects on the growth of
N. crassa. The vma-1 null strains showed clear pH
sensitivity, growing best at pH 5.5 and not at all at pH 7.0 or above.
Conditional lethality at basic pH has become a hallmark of
VMA deletion strains in S. cerevisiae (9, 10)
but, curiously, was not observed in A. gossypii (17). The
basis of the pH sensitivity is not understood. Growth of the N. crassa vma-1 null strains was strongly inhibited by the divalent
cation Zn2+ and weakly affected by Mn2+ and
Fe2+. We were surprised by the observation that 1 mM Cu2+ stimulated the radial expansion of
colonies on agar plates. By contrast, in S. cerevisiae,
Zn2+, Mn2+, and Fe2+ all strongly
inhibit growth of a VMA deletion strain, and
Cu2+ has given contradictory results (13, 14). Which
divalent cations (other than Ca2+) are stored in the
vacuole of N. crassa has not been reported, but these
results implicate the vacuole as a critical organelle for storage of
Zn2+. It was also important to observe that the pvn strains
were completely resistant to inhibition by concanamycin C, thus
strengthening our original finding that the V-ATPase is the cellular
target for this powerful antibiotic.
The most important consequence to the cell of inactivating the V-ATPase
may well be the effect on Ca2+ homeostasis.
Electrophysiological experiments have demonstrated that the vacuole of
N. crassa accumulates high concentrations of
Ca2+ (300 µM) and is largely responsible for
maintenance of low concentrations of Ca2+ in the cytosol
(0.1 µM) (31). High concentrations (100 or 200 mM) of Ca2+ in the medium restricted growth of
the N. crassa vma-1 null strains by approximately 50-60%
but also affected the wild type to a lesser extent (10-20%
inhibition). By comparison, high Ca2+ concentrations
strongly inhibit growth of VMA disruptant strains of
S. cerevisiae (11) and have little effect on the
VMA1 disruptant in A. gossypii (17). Preliminary
results of staining with chlortetracycline (CTC) suggested that
vacuolar Ca2+ stores were depleted in our vma-1
null strains.5 CTC fluoresces
when bound to Ca2+ in the vicinity of a membrane and is
suited to visualizing Ca2+ accumulated in vesicles and
organelles. It has been used to visualize Ca2+ gradients in
the growing hyphal tips of filamentous fungi (32). When we added CTC to
N. crassa cells, we saw brightly glowing hyphal tips in the
wild type and detected no fluorescence in the vma-1 null
strains. We have not yet attempted to measure Ca2+
concentrations in the cytosol of the vma-1 null mutants.
When growth was assessed as an increase in mass of an aerated liquid
culture, the N. crassa vma null strains behaved similarly to
VMA disruptants in S. cerevisiae and A. gossypii, i.e. growth was slower but not dramatically
different from the wild type. In either minimal or complete medium, the
two mutant strains of N. crassa had doubling rates of 1.3- and 1.8-fold those of the wild type. Similar to A. gossypii
and different from S. cerevisiae, N. crassa vma-1
null strains grew on non-fermentable substrates. Because N. crassa is an obligate aerobe, the mitochondria must be functional
in the vma null strains and not compromised as suggested by
the petite phenotype reported in early studies of VMA
deletion strains of S. cerevisiae (11).
Inactivation of vma-1 Has Striking Effects on Cell Morphogenesis in
N. crassa--
The most obvious and pronounced effects of inactivating
the vma-1 gene in N. crassa were on cellular
morphology and development. The primary effect appeared to be on the
control of tip elongation and hyphal branching. In the vma-1
null strains the rate of hyphal extension at the apex was greatly
decreased, and the frequency of branching was substantially increased.
This change could account for many of the observed growth phenotypes,
the slow progress of vma-1 null strains across an agar
surface, the thickening of the mycelial mat on an agar surface, the
formation of round balls in shaking liquid culture, and the failure of
mycelia to reach the surface in standing liquid. It might also restrict
the ability of the organism to form conidia, which differentiate on the
ends of aerial hyphae, to form perithecia, which differentiate from a
mass of mycelium, and to germinate from spores, where a tip must emerge
from the spore body. Although similar phenotypes have not been observed
in VMA disruptants of S. cerevisiae, a
temperature-sensitive mutant of VMA4 exhibited abnormal
morphologies including abnormally elongated or multiple buds (16).
Development was also affected in the VMA1 disruptant of
A. gossypii, where cells grew in pellets, which appeared
more dense and less branched, and failed to produce generative spores
(17).
How branching is controlled in filamentous fungi is not understood.
Normal tip growth involves a number of processes including cell wall
digestion, cytoplasmic streaming, and organellar distribution and
depends on an organized actin cytoskeleton (reviewed in Refs. 32-35).
Some of the agents that elicit increased branching affect the cell wall
or the cytoskeleton as follows: for example the sugar
L-sorbose and several fungicides, all inhibitors of
A logical deduction is that the primary effect on cell metabolism of
inactivating the V-ATPase is to raise the cytosolic free Ca2+, which results in the gross changes in morphogenesis.
Ca2+ normally accumulates in vacuoles of N. crassa and other fungi, and this sequestration depends on the
activity of the V-ATPase. In S. cerevisiae, deletion of a
V-ATPase gene was shown to increase cytosolic Ca2+
concentrations (11).
Vacuolar Morphology in vma-1 Null Strains Is Abnormal--
In
VMA deletion mutants of both S. cerevisiae and
A. gossypii the vacuoles had normal morphology. This was not
the case in N. crassa. Vacuoles in the vma-1 null
strains were highly variable when viewed under the light microscope.
Some were small and round like wild type organelles, but others were
large and distorted, frequently appearing as vacuoles within vacuoles.
This would suggest a role for the V-ATPase in determining organellar
structure within the endomembrane system, either direct or indirect via
Ca2+ and the cytoskeleton.
Phenotypes of pvn1 and pvn2 Are Not Identical--
In this work we
inactivated the vma-1 gene by RIPing, which introduces point
mutations into the genomic DNA sequence (28), rather than by gene
disruption as used in S. cerevisiae and A. gossypii. Fortuitously, we isolated strains with a heavily RIPed gene (pvn1, 42 point mutations) or a lightly RIPed gene (pvn2, 4 point
mutations). Both strains appeared to have non-functional V-ATPases. We
detected no concanamycin-sensitive ATPase activity in membranes from
the mutants, and both strains accumulated similar low levels of total
cellular arginine, an indicator of lack of vacuolar function in whole
cells. The two pvn strains had similar abnormalities in hyphal
morphology, in vacuolar morphology, and in responses to basic pH or inhibitors.
They differed strongly in one phenotype, spore viability. Spores of
pvn1 rarely germinated, demonstrating an important role of the V-ATPase
in this process. Indeed, the V-ATPase might be essential for
germination. Rare germlings may have resulted from inclusion of
maternal material with an active V-ATPase in the spore as happens with
mitochondria. Spores of pvn2 germinated with high efficiency. The two
strains showed small differences in two other properties. Pvn2 grew
slightly faster than pvn1 on agar plates or in liquid cultures, and
pvn2 arrested in meiosis at a later stage than pvn1. Because pvn2
probably makes a vma-1 protein product and pvn1 does not,
two explanations for the phenotypic differences seem plausible. First,
although we could not detect V-ATPase activity in pvn2, a very low
activity, below the sensitivity of our assay, could be present and
account for the observed differences. Second, the presence of the
protein product itself may be required for spore germination. Possibly,
subunit A of the V-ATPase has a second, unknown function in the cell.
Selective Suppression of the vma-1 Null Phenotype--
If wild
type N. crassa is grown in the presence of concanamycin, the
cells exhibit grossly distorted hyphal morphology and are unable to
grow at pH 7.2. We previously isolated concanamycin-resistant strains
that had mutated alleles of pma-1, the gene that encodes the
plasma membrane H+-ATPase (19). The pma-1
mutations suppressed the toxicity of concanamycin at basic pH but did
not suppress the morphological abnormalities. The mutations changed the
kinetic behavior of the enzyme, lowering the Km for
ATP from 1.2 mM in the wild type to 0.2-0.6 mM
in the mutants. We hypothesized that the concanamycin-resistant mutants
partially suppressed the sensitivity of N. crassa to
concanamycin because they could use ATP more efficiently to pump toxic
ions such as Ca2+ or H+ out of the cytoplasm
when the V-ATPase was not functional. We asked whether the ccr mutants
had a similar capacity to suppress the vma-1 null phenotype.
They did. A double mutant strain, containing ccr4-5 and pvn2, grew at
basic pH; other pvn phenotypes were not suppressed.
An unanticipated finding was that high concentrations of sorbitol
suppressed one of the morphological phenotypes of strain pvn2. On agar
plates supplemented with 1.0 M sorbitol, strain pvn2, but
not strain pvn1, produced conidia. We did not see effects on other
phenotypes, nor did we see conidiation in the presence of a high salt
concentration (1.0 M NaCl) or a 45 °C temperature-shock treatment.2 1.0 M sorbitol is likely to
desiccate the culture, and desiccation is one of the environmental
signals for inducing conidiation in N. crassa (43). Perhaps
sorbitol acts via the same pathway to induce conidiation in pvn2. The
simplest postulate for why pvn2 conidates and pvn1 does not is that
pvn2 does make a V-ATPase of very low activity and that the stress of
desiccation induces expression of the vma-1 gene and
synthesis of subunit A, resulting in sufficient V-ATPase activity to
allow conidiation.
Conclusion--
Inactivation of vma-1, the gene
encoding subunit A of the V-ATPase, has multiple effects on N. crassa. In particular, these effects implicate the V-ATPase as
involved in normal apical growth, perhaps regulating signals for
branching and differentiation. Our findings also implicate the V-ATPase
in the processes of germination and meiosis. As a relatively simple
eukaryote, which nevertheless shows considerable morphological
diversity, N. crassa is a good model organism to investigate
the participation of the V-ATPase in these complex phenomena.
We thank Dr. Robert Metzenberg of Stanford
University for providing strains of N. crassa. We are
grateful to Dr. N. B. Raju of Stanford University for the analysis
and interpretation of the meiotic process in asci of pvn strains. We
thank Dr. Axel Zeeck (University of Göttingen) and Dr. Karlheinz
Altendorf (University of Osnabrück) for their gifts of
concanamycin C.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
2
E. J. Bowman, unpublished results.
3
L. Taiz, University of California, Santa Cruz,
personal communication.
4
N. B. Raju, Stanford University, personal communication.
5
B. J. Bowman and E. J. Bowman,
unpublished results.
The abbreviations used are:
V-ATPase, vacuolar
proton-translocating ATPase;
CCC, concanamycin C;
HygR, resistant to hygromycin B;
HygS, sensitive to hygromycin B;
VM, Vogel's medium N;
ccr, concanamycin resistant;
pvn, putative vma
null;
CTC, chlortetracycline;
RIP, repeat-induced point.
Disruption of vma-1, the Gene Encoding the Catalytic
Subunit of the Vacuolar H+-ATPase, Causes Severe
Morphological Changes in Neurospora crassa*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as originally thought (15), the
deletion strains do not grow on non-fermentable substrates under usual
conditions and need inositol in the medium (11). Vacuoles in such
mutants are not acidified (9, 10), and cytosolic Ca2+
levels are elevated (11). Moreover, recent experiments with a
temperature-sensitive mutant of VMA4 show effects of
inactivation on polarized growth, bud formation, and cytokinesis
(16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N. crassa strains used in this study
progeny were tested on medium
supplemented with 0.2 mg/ml alanine or 1.5 mg/ml glycine; they grow on
alanine but not glycine plates. Supplements for tests of
Arg or His were 0.2 mg/ml arginine or
histidine. Resistance to hygromycin B (HygR) was scored as
the capacity to grow on medium containing 200 µg of hygromycin B/ml,
added before autoclaving. The HygR phenotype was scored
more easily in liquid medium than on agar plates. Resistance to
concanamycin C was scored as the capacity to grow on agar plates with
VM/sucrose medium containing 0.2 µM CCC, added after
autoclaving, at pH 7.2 for non-pvn strains or at pH 5.8 for pvn strains.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and has a
RIPed vma-1 gene. It also contains a functional
vma-1 gene, introduced on a plasmid with an hph
gene, which confers resistance to hygromycin, on a different,
unidentified chromosome. Strain vma-1RR1a was crossed to the wild type
strain 74A (Table II, Cross 1). Mature,
germinated spores were picked to liquid medium. At first all spores
that germinated and grew appeared to be Am+ (indicating a
normal endogenous vma-1 gene) or HygR
(indicating a functional ectopic vma-1 gene). However, three isolates grew very slowly. Growth tests indicated they were
Am and HygS, the expected phenotype of a
strain with a RIPed endogenous vma-1 gene. We named these
slow-growing strains pvn1 (for putative
vma-1 null).
Generation of a vma-1 null strain by recovery of vma-1RIP from
the RIP/Rescue strain
(Table II,
Cross 2). Many spores failed to germinate. Among spores that did
germinate, 306 were Am+ and grew fast like wild type, and 7 were Am and grew slowly like pvn1. Thus, slow growth
cosegregated with am and the adjacent
vma-1 locus. The low recovery of pvn1 progeny and the high
number of ungerminated spores further suggested to us that the mutation
resulted in a low rate of germination, approximately 1% if we assume
that most of the ungerminated spores had the pvn1 genotype.
progeny, predicted to be
vma-1RIP, were all HygR, thus able to
grow like wild type when a functional ectopic copy of vma-1
was present. Twelve additional progeny, not included in Table II, were
the result of crossover events between am and inl
loci; again, strains with the vma-1RIP gene could
grow when the functional ectopic copy was present. In this particular
experiment none of the 86 isolates displayed the pvn1 phenotype,
presumably because of the low germination rate of pvn1 spores.
Rescue of pvn1 phenotype by an exogenous copy of the vma-1+
gene
View larger version (40K):
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Fig. 1.
pvn strains contain a single copy of the
vma-1 gene, which is mutated. Southern blots of
genomic DNA were digested with BamHI and probed with
vma-1 as described under "Experimental Procedures."
A, strain pvn1 (pvn1-121A) was derived from a cross between
the wild type 74A and the RIP/Rescue strain vma-1RR1a, which has a
non-functional copy of vma-1 tightly linked to
am132 at the endogenous location on linkage
group V and a second functional copy of the vma-1 gene
inserted with an hph marker at an ectopic location. Strain
pvn1 has a single copy of vma-1 at the endogenous
vma-1 locus. B, strain pvn2 (pvn2-53-19A) was
derived from a cross between arg12 and bump,
which has a normal vma-1 gene at the endogenous location and
a partial vma-1 gene inserted with an hph marker
and tightly linked to arg12 on linkage group II. Strain pvn2
has a single copy of vma-1 at the endogenous locus. DNA
corresponding to the endogenous vma-1 gene in strains pvn1
and pvn2 was obtained by polymerase chain reaction, subcloned, and
sequenced (see "Experimental Procedures"). Both clones contained
multiple single base changes, a result of mutations caused by RIP.
Positions of mutations are indicated in diagrams.
C, strain pvn1, 42 mutations; D, strain pvn2, 4 mutations.
on chromosome V, and it was rescued by a
functional ectopic copy of vma-1, and the sequence of the endogenous
vma-1 gene had multiple point mutations. The strain was
characterized by particularly slow growth in liquid medium and a very
low efficiency of germination in sexual spores. These properties likely
explain our earlier failure to obtain such strains (8).
strain
(Table IV, Cross 1). Fifty progeny had
the Arg12 phenotype, 43 had the bump phenotype, and 3 were Arg, slow-growing strains reminiscent of pvn1
mutants in phenotype. We conducted further tests to determine if they
were vma-1 mutants. In two strains, the slow growth
phenotype was not linked to either am or arg12.
However, the third strain showed clear linkage between the slow growth
phenotype and am (Table IV, Cross 2) and exhibited the
characteristic morphology of pvn1 on an agar plate, making it a
promising candidate for a second vma-1 null strain. It was named pvn2. A genetic cross demonstrated that the pvn2 phenotype could
be rescued by a functional, ectopic copy of
vma-1+ (Table IV, Cross 3). The cross was
potentially confusing because the two pvn strains had different alleles
of vma-1RIP. The inheritance of one or the other
allele was inferred by scoring the closely linked am locus;
vma-1RIP(1) in pvn1 was linked to
am, and vma-1RIP(2) in pvn2
was linked to am+. HygR
served as the marker for the functional, ectopic
vma-1+ gene. As before, pvn1 progeny grew only
when the ectopic vma-1+ gene was present (22 isolates). pvn2 progeny grew like wild type in the presence of the
ectopic vma-1+ gene (15 isolates), indicating
rescue, and pvn2 progeny germinated and grew slowly in the absence of
the ectopic vma-1+ gene (17 isolates). Thus, in
contrast to ascospores of strain pvn1, ascospores of pvn2 showed no
impairment in germination (Table III, Crosses 2 and 3).
Generation of strain pvn2 by RIP
subunits of F-ATPases. Only one, G170S, is
conserved in both vacuolar and the related archaebacterial ATPases.
This is an interesting residue because of its location within the
"non-homologous" region of V-ATPases, the region which has no
counterpart in F1. The function of this region in
V-ATPases remains unknown; however, its deletion from VMA1
in S. cerevisiae appeared to be
lethal.3
View larger version (54K):
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Fig. 2.
Pvn strains are sensitive to elevated pH and
resistant to concanamycin. A, wild type (74A) and the
two pvn strains (pvn1-121A and pvn2-53-19A) were grown at 30 °C on
agar plates containing minimal medium, adjusted as indicated in the
figure. B, wild type and the two pvn strains were grown on a
reciprocal shaker for 2 days at 25 °C in liquid minimal medium
supplemented with either 20 mM 3,3'-dimethyl glutaric acid
(pH 3.5 and 4.5) or 20 mM HEPES (pH 5.5-7.5) and adjusted
to the indicated pH with NaOH. Cells were collected on a filter, rinsed
with distilled water, lyophilized, and weighed.
Growth rates of pvn mutants
Effect of selected compounds on growth of pvn strains
View larger version (59K):
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Fig. 3.
Inactivation of vma-1 has
dramatic effects on hyphal morphology, restricting extension and
stimulating branching. Wild type (74A) and the two pvn strains
(pvn1-121A and pvn2-53-19A) were grown 24 h at 30 °C on agar
plates containing minimal medium at pH 5.8. Bar, 100 µM.
View larger version (67K):
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Fig. 4.
Inactivation of vma-1 alters
vacuolar morphology. Wild type (74A) and the two pvn strains
(pvn1-121A and pvn2-53-19A) were grown overnight at 30 °C in
standing liquid medium and transferred to a microscope slide. Sections
of mycelium were photographed under Nomarski optics at × 1000 magnification. Bar, 4 µm.
View larger version (71K):
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Fig. 5.
The double mutant strain, pvn2 ccr4-5, grows
at alkaline pH. Wild type (74A), strain pvn2-53-19A, and the
double mutant strain of pvn2, ccr4-5a were grown at 30 °C on agar
plates containing minimal medium supplemented with 20 mM
HEPES base, adjusted to pH 5.8 to 7.8 with NaOH for 1 day (74A) or 4 days (mutants).
View larger version (120K):
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Fig. 6.
1.0 M sorbitol induces
conidiation in strain pvn2. Strains pvn1 and pvn2 were grown for 5 days at 30 °C followed by 7 days at room temperature on agar plates
containing minimal medium with or without the addition of 1.0 M sorbitol. Abundant conidia were produced on the pvn2
strain grown on sorbitol but not on the pvn1 strain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1-3-glucan synthetase (35); and cytochalasin A (36), an antiactin
drug. Other agents appear to disrupt calcium homeostasis as follows: for example A23187, a Ca2+ ionophore (37); verapamil, a
Ca2+ channel blocker (38); and cyclosporin A, an inhibitor
of calcineurin, a Ca2+/calmodulin-dependent
protein phosphatase (39). In addition, colonial mutants characterized
by profuse branching are plentiful in fungal collections, but only a
few have known genetic deficiencies. In N. crassa these
include the following: crisp, deficient in adenyl cyclase (40);
NKIN-deficient strain, a kinesin null mutant (41); cot-1, altered in a
cAMP-dependent protein kinase (42); and pvn1 and pvn2
described in this paper. A common denominator for many of these
observations is cytosolic free Ca2+ concentration. A tip
high cytosolic free Ca2+ gradient has often been cited as a
primary factor in regulating hyphal extension and branching in fungi.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biology,
University of California, Santa Cruz, CA 95064. Tel.: 831-459-2245; Fax: 831-459-3139; E-mail: rbowman@biology.ucsc.edu.
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RESULTS
DISCUSSION
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