FEMS Microbiology Letters 108 (1993) 7-10
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
FEMSLE 05330
Carnitine acetyltransferase is absent
from acuJ mutants of Aspergillus nidulans
M. M i d g l e y
Department of Applied Biology, University of Hull, Hull, UK
(Received 7 December 1992; revision received 4 January 1993; accepted 4 January 1993)
Abstract." The acuJ mutant of AspergiUus nidulans has been shown to lack carnitine acetyltransferase (CAT) activity when grown
under conditions where this activity is readily detectable in wild-type strains. Revertants selected for growth on acetate recover
CAT activity and the ability to grow on long-chain fatty acids. When growing on carbon sources such as sucrose, cytosolic acetyl
coenzyme A was generated by adenosine triphosphate (ATP): citrate lyase.
Key words: Aspergillus nidulans; Carnitine acetyltransferase; Acetyl-coenzyme A
Introduction
An earlier investigation of acetate metabolism
in Aspergillus nidulans led to the isolation of a
series of mutants blocked in acetate catabolism
[1]. In many cases the enzyme specified by a
particular complementation group was identified
by direct assay but in a number of mutants the
defect was not ascertained. Mutants designated
acuC ( facC ), acuH, acuL and acuJ carry unidentified lesions in acetate metabolism. The latter
three mutants are also defective in butyrate
catabolism [1].
Growth on these carbon sources, and also
long-chain fatty acids, requires that acetyl groups
are effectively transported between different cellular compartments. Studies with yeasts such as
Candida tropicalis and Saccharomyces cerevisiae
have indicated a major way in which this is
achieved is by the enzyme carnitine acetyltransferase (CAT, EC 2.3.1.7) which, in a readily reversible reaction, catalyses the formation of
acetylcarnitine from acetyl-CoA and carnitine [24]. Transfer of the acetylcarnitine between major
cell compartments such as mitochondria, peroxisomes and cytosol can then take place and acetylCoA can be regenerated by a suitably located
CAT.
The findings presented in this report indicate
that CAT activity is absent in strains of A. nidulans carrying the mutation designated acuJ.
Materials and Methods
Strains of A. nidulans
Correspondence to: M. Midgley, Department of Applied Biology, University of Hull, Hull, HU6 7RX, UK.
The origin of the strains used in this study has
been described by Armitt et al. [1]. Thus R21
(yA2pabaA1) is the parent of acuJ211, 213, 214,
216, 256 and 257, acuH253 and acuL217 and
R153 (wA3pyroA4) is the parent strain of acuJ
302, 335, 343, 346, 349 and 351. The acuC strain
used was pabaA1 acrA1 sD85 acuCl02, and the
strain deficient in acetyl-CoA synthetase was
H902 (yA2 pabaA1 acuA204). Organisms were
obtained from Dr. C. Roberts (University of Leicester), Dr. H. Sealy-Lewis (University of Hull,
Hull), and the Fungal Genetic Stock Center
(Kansas).
Media, growth conditions and genetic techniques
The defined medium of Pontecorvo [5] was
used throughout with appropriate supplements.
Conidia (5 x 106 final concentration) were used
as inocula and cultures were grown with shaking
at 30°C. Carbon sources used were sucrose (100
mM), s u c r o s e / a c e t a t e (20 m M / 1 0 0 mM) and
s u c r o s e / T w e e n 80 (20 m M / 1 2 g / l ) and the nitrogen source was 10 mM urea. Cultures were
generally grown for 20 h. Standard genetic techniques were used [5,6]. Revertants of acuJ211
designated S1, $7 and $9 were isolated after mild
UV mutagenesis ( < 5% kill) by plating conidia on
minimal acetate medium. In back-crosses to
wild-type at least 100 progeny were scored for
acetate utilization.
Results and Discussion
A preliminary survey of CAT activity in the
mutants of interest was carried out using extracts
prepared from organisms grown for 20 h on a
mixture of sucrose and acetate. CAT activity was
detected in all but the strain carrying the acuJ211
mutation. The specific activities, expressed as
n m o l - t (mg protein)-1 observed in two independent extracts were: wild-type (R21), 55 and 53;
acuL217, 58 and 56; acuH253, 67 and 68;
acuCl02, 30 and 25. Assays of isocitrate lyase
(ICL) activity indicated that acetate had acted as
an effective inducer under these growth conditions except for the acuC strain which showed
only weak induction (data not shown).
These observations were extended to other
members of the acuJ complementation group,
grown on either sucrose, sucrose plus acetate or
sucrose plus Tween 80 (Table 1). Tween 80 ( ~
Table 1
S p e c i f i c a c t i v i t y o f C A T a n d I C L in w i l d - t y p e a n d acuJ s t r a i n s
o f A. nidulans g r o w n o n d i f f e r e n t c a r b o n s o u r c e s
Organism
Specific activity
CAT
ICL
A
R21
Preparation of cell extracts and enzyme assay
Biomass was collected by filtration through
nylon gauze (mesh size 100 /zm), washed briefly
with distilled water and then pressed between
absorbent tissues. The resulting cell-cake was resuspended in either 50 mM Tris. HCI pH 7.5, or,
if ATP:citrate lyase was to be assayed, in 20%
w / v glycerol in 50 mM imidizole/HCl buffer (pH
7.8) containing 5 mM mercaptoethanol and 1 mM
E D T A [7]. Cell disruption was achieved by two
passages through a pre-cooled French pressure
cell at 35 MPa. The supernatant obtained after
centrifugation at 2000 x g for 10 min was assayed
for appropriate enzymes as previously described
[1,8] with the addition of 5 mM NaN 3 to
ATP:citrate lyase assays to minimise N A D H oxidase activity. All specific activities are expressed
as nmol min-1 (mg protein)-1.
Growth
substrate
17
17
-
-
55
53
89
84
86
81
19
13
-
-
-
-
59
63
65
83
80
sucrose
sucrose/Tween
80
-
sucrose
.
sucrose/acetate
acuJ351
.
.
-
69
66
-
-
258
245
sucrose
19
24
-
-
sucrose/acetate
61
66
67
79
57
10
sucrose/Tween
acuJ302
.
-
sucrose/Tween
R153
B
sucrose/acetate
sucrose/acetate
acuJ256
A
sucrose
sucrose/Tween
acuJ211
B
80
53
7
sucrose
-
-
-
sucrose/acetate
-
162
164
35
.
32
sucrose/Tween
sucrose
80
80
sucrose/acetate
sucrose/Tween
80
.
.
.
-
-
146
148
-
-
45
60
S p e c i f i c a c t i v i t i e s [ n m o l m i n - 1 ( m g p r o t e i n ) - 1 ] a r e s h o w n in
columns A and B for two independent
i n g acuJ m u t a n t s
described
in t h e
extracts. The remainMaterials
and
Methods
s e c t i o n w e r e a l s o s h o w n to h a v e n o C A T a c t i v i t y w h e n g r o w n
on sucrose. -, not detected [ < 2 nmol min-1 (mg protein)-1].
Table 2
Specific activities of CAT and ICL in wild-type and acetate
positive acuJ revertants of A. nidulans
Organism
R21
S1
$7
$9
Growth
Substrate
sucrose
acetate
Tween 80
sucrose
acetate
Tween 80
sucrose
acetate
Tween 80
sucrose
acetate
Tween 80
Specificactivity
CAT
ICL
A
B
A
B
17
832
532
22
310
165
21
232
190
30
620
802
17
630
503
25
236
171
22
244
199
28
434
605
100
175
144
40
44
30
98
23
109
160
118
45
63
31
105
17
Specific activities [nmol min-1 (mg protein)-1] a r e shown in
columns A and B for two independent extracts. Cultures were
grown on acetate (100 mM) or Tween 80 (12 g/l) for 40 h. -,
not detected [ < 2 nmol min -I (mg protein)-1].
70% polyethoxyelthylenesorbitan monooleate)
acted as a good carbon source for wild-type A.
nidulans, and was used as an alternative to butyrate, a relatively poor carbon source. By analogy with other fungi it is presumed that the
utilization of Tween is brought about by the
action of a lipase, the fatty acids liberated being
further catabolised by a peroxisomal /3-oxidation
system producing acetyl-CoA [9]. As shown, significant induction of ICL, a peroxisomal enzyme,
was observed in the presence of acetate and
Tween 80.
CAT activity was readily detected in extracts
of wild-type strains even when grown on sucrose
but was not detected in the acuJ strains under
any of the growth conditions used. Extracts prepared from R21 grown on acetate or Tween 80
for 40 h, in the absence of sucrose, gave higher
specific activities (Table 2). The catabolite repression effects by sucrose reported in Table 1 are
well documented in A. nidulans [10].
Although the cellular location of the CAT
activity detected in sucrose-grown cells has not
been established it is unlikely to be peroxisomal
since such organelles are not known to play a
major role in growth on this carbon source. In
some yeasts it has been suggested that CAT activity plays a role in generating cytosolic acetyl CoA
using acetyl-carnitine exported from mitochondria [11]. Strains carrying the acuJ mutation show
no defect in growth on carbon sources such as
sucrose suggesting that the generation of cytosolic acetyl-CoA by this route was not obligatory in
A. nidulans and was met by the enzyme
A T P : c i t r a t e lyase (ACL), an enzyme readily detectable in cell-extracts of acuJ mutants and
wild-type. The following specific activities were
measured in extracts prepared from sucrosegrown R21, acuJ211 and acu256: 122, 134 and
105 respectively (mean of two independent values). Similar values were recorded for sucrosegrown H902 (acetyl-CoA synthetase deficient)
suggesting that acetyl-CoA synthetase plays no
obligatory role in generating cytosolic acetyl-CoA
when A. nidulans grows on sugars.
Revertants of acuJ211, selected for growth on
acetate, also regained the ability to grow on
Tween 80. Measurement of CAT activity in cellextracts prepared from three independent revertants grown on sucrose, acetate or Tween 80
indicated that CAT activity was regained (Table
2). When such revertants were back-crossed to
the wild-type no acetate-negative recombinants
were detected suggesting the original mutation
had reverted rather than the presence of a suppressor mutation.
These findings suggest that in A. nidulans CAT
plays a major metabolic role when there is a
requirement for increased intracellular transport
of acetyl groups. Under such conditions (e.g.
growth on acetate or fatty acids) CAT activity is
required in multiple cell compartments. Mutations at a single locus lead to the loss of all CAT
activity. This observation is consistent with the
existence of a single structural gene producing an
enzyme targetted to different locations or a regulatory mutation preventing expression of multiple
structural genes. Work with alkane grown C. tropicalis is consistent with the first possibility, i.e. a
single structural gene with differential processing
[4]. Whether this is true for A. nidulans remains
to be established.
10
Acknowledgements
I t h a n k Dr. C. R o b e r t s (Leicester), Dr. H.
Sealy-Lewis (Hull) a n d Steve D a c k for the generous supply of strains, genetic expertise a n d skilled
technical assistance respectively.
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