Download An acetate-sensitive mutant of Neurospora crassa deficient in acetyl

Journal of General Microbwlogy (1 992), 138, 1797-1 800. Printed in Great Britain
1797
An acetate-sensitive mutant of Neurospora crassa deficient in
acetyl-CoA hydrolase
I. F. CONNERTON,~*
W. MCCULLOUGH~
and J. R. S. FINCHAM3
Protein Engineering Department, AFRC Institute of Food Research, Earley Gate, Whiteknights Road,
Reading RG6 2EF, UK
Department of Biological and Biomedical Sciences, University of Ulster at Jordanstown, Newtownabbey,
Co. Antrim BT37 OQB, UK
Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
(Received 21 January 1992; revised 22 April 1992; accepted 18 June 1992)
The predicted amino acid sequence of the product of the acetate-inducible mu-8 gene of Neurospora crassa,
previously of unknown function, has close homology to the recently published sequence of Sacchuomyces
cereuisiae acetyl-CoAhydrolase. An acu-8 mutant strain, previously characterized as acetate non-utilizing,shows
strong growth-inhibitionby acetate, but will use it as carbon source at low concentrations.The mutant was shown
to be deficient in acetyl-CoA hydrolase and to accumulate acetyl-CoA when supplied with acetate. As in
Saccharomyces, the Neurospora enzyme is acetate-inducible.
Introduction
The Neurospora crassa acu-8 gene is one of three cloned
by Thomas et al. (1988) on the basis of their preferential
transcription when sucrose was replaced by acetate as
carbon source. The two others were found to code for
acetyl-CoA synthetase and malate synthase respectively,
but the function of acu-8 has remained obscure. Marathe
et al. (1990) sequenced the gene, and by making use of the
RIP (repeat induced point mutation) phenomenon
(Selker et al., 1987)obtained a mutant strain in which the
acu-8 sequence was extensively disrupted. The effect of
the RIP phenomenon in Neurospora is to cause premeiotic disruption of duplicated sequences in the same
nucleus of specialized dikaryotic cells. Transformed and
resident sequences therefore participating in this process
become heavily cytosine-methylated and the subjects of
extensive GC-AT base transitions. The resulting acu-8
mutant, as expected, failed to grow on 40 mwacetate as
carbon source (the concentration optimal for the wildtype), but, surprisingly, was able to use ethanol. The
mutant was found to be normal in its ability to take up
acetate from the growth medium.
The sequence of acu-8 includes an open reading frame
of 521 codons interrupted by two introns. Initially, it was
not found to resemble any other published sequence, but
* Author for correspondence.Tel. (0734)357000;fax (0734)267917;
e-mail, CONNERT0NBAFRC.ARCB.
the recently published sequence of Saccharomyces cereuisiae glucose-repressible acetyl-CoA hydrolase (Lee et al.,
1990) shows close homology with acu-8. Lee et al. (1990)
suggested that the function of acetyl-CoA hydrolase in
yeast was to control the level of acetyl-CoA, which might
otherwise become toxic under conditions of glucose
derepression. Acetate toxicity seemed a likely explanation of the failure of the acu-8 mutant to grow on acetate.
This communication provides supporting evidence for
this hypothesis.
Methods
Fungal culture. Neurospora crassa was grown in liquid medium as
previously described (Thomas et al., 1988). Acetate was added from a
stock solution of 1 M-acetic acid adjusted to pH 5.2 with NaOH.
Acetyl-CoA hydrolase assay. Extracts were made by grinding
mycelium with sand in 0.4M-sucrose, 10 mM-Tricine, pH 7.5, and
centrifuging successively at 3000 g for 10 min to remove the cell debris
and at 23500g for 30 min to pellet the membrane fraction at 4 "C.
Malate synthase, which would otherwise interfere with the assay, was
predominantly in the pellet. Acetyl-CoA hydrolase, which remained in
the supernatant, was assayed in a reaction mixture containing 50 m ~ potassium phosphate, pH 7-2, and 0.2 mM-acetyl-CoA at 30 "C.
Hydrolysisof acetyl-CoA was assayed by following the appearance of
free -SH groups, measured by addition of dithiobis(2-nitrobenzoic
acid) and determining the increase in absorbance at 412 nm. AcetylCoA synthetase was assayed as described previously (Thomas et al.,
1988).
0001-7355O 1992 SGM
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1798
I. F . Connerton, W . McCullough and J . R . S . Fincham
1 MASPIASAALRAR-QGPSMLKKLCNPEDMLQHFPNGAYIGWSGFTASATP 49
I
I I I I
Ill
I II I IIIIII
II
1 M-T-I-SNLLKQRVRYAPYLKKVKEAHELIPLFKNGQYLGWSGFTGVGTP 47
50 R-RSY Y LADHVEKNASRPAQVQPLRRCLRRRRDRERWAALDMIAHDRRRA 98
I IIIIII
I
Ill Ill
II
48 K A V P E A L I D E W E X N N L Q G K L R F N L F V G A S A G P E E N R W A E H 94
99 PHQVGKNIAKGINEGRINFFDKHLSMFPVDLVYGYYTKDR-QNKJSLDWC
IIIIII Ill II Ill IIIIIIIIII II II II I II II
95 PHQVGKPIAKAINQGRIEFFDKHLSMFPQDLTYGFYTFGRKDNKILDYTI
147
144
148 VEATEIKEDGSIVLGASVGATPELIQMADKVIIEVNTAIPSFDGLHDITF 197
Ill IIIIIIII I I l l
II I
IIIIIIIIII Ill I Ill
145 IEATAIKEDGSIVPGPSVGGSPEFITVSDKVIIEVNTATPSFEGIHDIDM 194
198 SDLPPNRKPYLIQQCRDRIGTTSVPVDPEWGIIECTTPDQTLPNSPAD 247
II IIII
I I I IIIIIIII I I I II II I I
195 PVNPPFRKPYPYLKVDDKCGVDSIPVDPEWAIVESTMRDQVPPNTPSIJ 244
248 ETATAIAGHLIEFFEHEVAHGRLPKNLLPLQSGIGNIANAVIGGLETSNF 297
IIIIII Ill I I IIIII IIIIIIIIIIIIIIIII II
I
245 DMSRAIAGHLVEFFRNEWKHGRLPENLLPLQSGIGNIANAVIEGLAGAQF 294
298 KNLNVWTEVIQDTFLDLFDSGKLDFATATATSIRE'SPTGFERF'YKNWDNYYD347
I I IIIII II IIII I II IIII I
I l l
I l l
295 KHLTVWTEVLQDSLLDLFENGSLDYSTATSVRLTEKGFDWANWEXFKH 344
348 KLLLRSQSVSNAPEIIRRLGVIGMNTPVEVDIYAHANSTNVMGSRMLNGL 397
I IIII Ill II Ill II IIIIIIIIIIIIIIIIII IIIIIIII
345 RLCLRSQWSNNPEMIRRFPVIAMNTPVEVDIYAHANSTNVNGSRMLNGL394
398 GGSADFLRNSKYSIMHTPSTRPSKTDAHGVSCIVPMCTHVDQTEHDLDVI 447
IIIIIIIII I IIII II II I I I I Ill1 IIIIIIIIII
395 GGSADFLRNAKLSIMHAPSATKVDFTGISTIVPMASHVDQTEHDLDIL 444
448 VTENGLADVRGLSPRERARVIIDKCAHDVYKFILKAYFEKAEFECLRKGM497
I I I I I I I I I I I I I I I I1 I I I I I
I I
II
445 VTDQGLADLRGLSPKERAREIINKCAHPDYQALLTDYLDRAEHYAKKHNC494
498 GHEPHLLFNSFDMHKALVEEGSMAKVW ..... 525
IIII I I I I I I I I II
495 LHEPHMLKNAFKFHTNLAEKGTM-KVDSWEFVD 526
Fig. 1. Comparison of the predicted amino acid sequences of the N. crassa acu-8 product (upper line) and S.cereuisiue acetyl-coenzyme
A hydrolase (lower line). Vertical lines indicate identical amino acid residues and horizontal lines indicate gaps inserted to maximize
homology. These sequences appear in the PIR database under the accession numbers A35195 and A363316 for S. cereuisiae and N .
crassa respectively.
[l3C]Acetutefeedingexperiments. The fate of [l3C1acetate fed to the
was investigated by NMR in perchloric acid extracts
essentially as described by Thomas & Baxter (1987).
U C U - ~mutant
Database searches and sequence alignments. These were done using the
UWGCG suite of programs of Devereux et a/.(1984), including the FASTA
program of Pearson & Lipman (1988).
Results and Discussion
Fig. 1 shows the comparison of the predicted amino acid
sequences of yeast acetyl-CoA hydrolase and the N .
crassa acu-8 product. The two sequences show 61 %
amino acid identity. There is significant similarity Over
virtually the entire sequences, with the greatest differences near to the N- and C-termini.
The acu-8 mutant is apparently totally deficient in
acetyl-CoA hydrolase (Table l), as would be predicted
from the extensive modifications made in the gene by the
RIP process (Marathe et al., 1990). The Neurospora
enzyme resembles that from yeast in being repressed on
sucrose as carbon source and derepressed on acetate.
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Acetyl-CoA hydrolase mutant of Neurospora
Table 1. Acetyl-CoA synthetase and hydrolase actiuities in
N . crassa wild-type and the acu-8 mutant
The activities recorded are means of duplicate assays and are
representative of three independent determinations.
Specific activity
[nmol min-*
(mg protein)-']
Carbon
source
Strain
W ild-type
(74'4)
acu-8
Synthetase
Hydrolase
4
<1
Sucrose
Sucrose then 8 h
Sucrose then 8 h
Sucrose
Sucrose then 8 h
Sucrose then 8 h
glycerol
acetate
7
92
1
5
85
glycerol
acetate
11
36
<I
<I
(1
Table 2. Growth of N . crassa wild-type and acu mutants on
diflerent acetate concentrations
Standing liquid cultures (15 ml) were each inoculated with about
lo6 conidia. Values for growth are means of duplicates. -, Already
harvested; tr., trace (visible fuzz of germinated spores not
recoverable as weighable mycelium); ND, not detectable.
Growth (mg dry wt) on:
Acetate
Strain
Wild-type
(74'4)
acu-8
acu-3
acu-5
Incubation
time (h)
Sucrose
13-3m~
4
8
16
40m~
48
31.4
1.0
2-1
4.3
7-1
48
83
125
48
125
33.3
tr.
0.8
tr.
1.1
ND
-
ND
ND
-
-
-
tr.
4.0
23-1
ND
ND
ND
ND
ND
ND
ND
ND
48
125
36.1
ND
ND
ND
ND
ND
ND
ND
ND
-
tr.
The results of a representative experiment on growth
of the acu-8 mutant on acetate as carbon source are
shown in Table 2. On concentrations of acetate below
5 mM, acu-8 grew only slightly less well than the wildtype. At higher concentrations growth was delayed, and
at 4 0 m ~ the
, concentration hitherto used for growth
tests because it is near-optimal for the wild-type, the
mutant showed only a trace of growth even after 5 d at
30 "C. These findings explain the ability of the mutant to
grow on ethanol (Marathe et al., 1990), which presumably provides a sustained supply of acetate at low
concentrations.
Other experiments (data not presented here) showed
that growth on 0.5 M-glycerol, a poor carbon source used
by acu-8 as well as by the wild-type, was strongly
inhibited by 5 mw-acetate in acu-8 but enhanced by the
same acetate concentration in the wild-type. Two other
acetate non-utilizing mutants, acu-3 and acu-5, respec-
1799
tively defective in isocitrate lyase and acetyl-CoA
synthetase, were also inhibited by acetate when grown on
glycerol as carbon source. As shown in Table 2, they
differed from acu-8 in showing no growth on acetate
alone at any concentration.
NMR analysis showed that acu-8 mycelium did not
accumulate [ 3C]acetate after transfer from sucrose to
40mM-acetate as carbon source, but it did accumulate
acetyl-CoA. Unlike the wild-type, acu-8 mycelium
exhibited no significant flux through the glyoxylate cycle
10 h after transfer to 40 mM acetate.
These results are entirely consistent with the conjecture of Lee et al. (1990) that acetyl-CoA hydrolase
performs the function of preventing a toxic build-up of
acetyl-CoA under conditions of carbon catabolite derepression. It seems unlikely that it is involved in export
of acetyl groups across the mitochondrial membrane, as
discussed by Kohlhaw & Tan-Wilson (1977), because, as
noted above, it appears to be cytosolic and not associated
with the mitochondrial fraction.
One may ask how the cytosolic location of acetyl-CoA
hydrolase is compatible with the maintenance of an
adequate supply of acetyl-CoA to the mitochondria and
glyoxysomes. It may be that the organelles can assimilate
acetyl-CoA at high efficiency from a steady-state
cytosolic concentration that is well below the Michaelis
constant of the hydrolase, estimated by Kohlhaw & TanWilson (1977) as 2.2 x 1 0 - 4 ~
for the yeast enzyme.
Acetyl-CoA hydrolase may be a safety-valve which, even
though necessary, is most of the time hardly used.
An additional possible reason why sufficient active
acetate survives transit through the cytosol is that it is
stored in some energetically equivalent derivative not
subject to cytosolic enzymic degradation. Two alternative protected derivatives discussed by Kohlhaw & TanWilson (1977) are citrate, through which acetate could
cycle via citrate synthetase and ATP :citrate lyase, and
acetylcarnitine interchangeable with acetyl-CoA
through carnit ine-CoA acetyltransferase. These authors
suggested that the latter enzyme, which they found to
have a much higher affinity than acetyl-CoA hydrolase
for acetyl-CoA, may be instrumental in the export of
acetyl groups through the mitochondrial membrane.
Both ATP :citrate lyase and carnitine-CoA acetyltransferase have been shown to be present in fungi (Ratledge
&Gilbert, 1985; Jernejc et al., 1991),and the acetyltransferase has been demonstrated in Neurospora (I. F.
Connerton, unpublished result). In the n-alkane-utilizing
yeast Candida tropicalis, two forms of the carnitine
acetyltransferase have been reported, one in the mitochondria and one in the peroxisomes, which fulfil the
function of glyoxysomes in this organism (Ueda et al.,
1982). It will be interesting to see whether similar
isoenzymes exist in Neurospora.
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1800
I . F. Connerton, W . McCullough and J . R. S. Fincham
We thank the SERC for support for I. F. C. and for making available
NMR facilities. We also thank Dr John Archer for helpful discussion.
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