YEAST
VOL.
8 193-203 (1992)
Characterization of Mutants of the Yeast Yarrowia ZipoZ’tica Defective in Acetyl-Coenzyme A Synthetase MARIAN KUJAU, HERBERT WEBER AND GEROLD BARTH*t Institute for Microbiology and Experimental Therapy, Jena, Beutenbergstr. II,D-6900 Jena, Germany Received 22 June 1991
The expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl-coenzyme A, which is produced by acetyl-coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate-non-utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In ic12 mutants, lack of ACS activity resulted in a non-induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the structural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICLI, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPRI =glyoxylate pathway regulation) was detected in which trans-dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source. KEY WORDS -Glyoxylate
pathway; acetyl-coenzyme A synthetase; isocitrate lyase; dominant mutations; Yarrowia
lipolytica.
INTRODUCTION The glyoxylate pathway has to replenish intermediates (succinate, malate) of the tricarboxylic acid cycle during growth on C, compounds or fatty acids for biosynthesis of gluconeogenetic compounds and amino acids (Kornberg, 1966). Furthermore, biosynthesis of glycine, from which heme and serine are formed, starts from glyoxylate during growth on such substrates (Figure 1). The main enzymes of this pathway are isocitrate lyase (ICL), which converts threo-D-isocitrate into glyoxylate and succinate, and malate synthase (MAS), which synthesizes malate from glyoxylate and acetyle-coenzyme A. However, during growth on glucose, serine is formed from 3-P-glycerate via 3-P-hydroxypyruvate and ophosphorserine, and glycine is synthesized from serine. Oxaloacetate arises by other anaplerotic pathways from phosphoenolpyruvate or pyruvate on glycolytic substrates. Therefore, the syntheses of ICL and MAS are repressed during growth on hexoses. *Present address: Department of Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland. tAddressee for correspondence. 0749%503X/92/030193-11 $05.50 1992 by John Wiley & Sons Ltd
0
Acetyl-coenzyme A, which is formed by acetylcoenzyme A synthetase (ACS) from acetate or by thiolase from fatty acids during P-oxidation, induces the synthesis of ICL and MAS during growth on these carbon sources. Mutants deficient in ACS activity are only known from three filamentous fungi. Such mutants were identified on the basis of fluoroacetate resistance in Aspergillus nidulans (Apirion, 1965) and selected among acetate-nonutilizing mutants (Acu-) of Neurospora crassa (Flavell and Fincham, 1968a,b) and Coprinus cinereus (Casselton and Casselton, 1974). Recently, the ACS-encoding genes of the ascomycetous fungi A . nidulans and N . crassa were cloned and sequenced (Thomas et al., 1988; Sandeman and Hynes, 1989; Connerton et al., 1990). Fluoroacetate-resistant mutants CfacB)of A . nidulans were isolated in which loss-of-function inhibits induction of ACS and of glyoxylate cycle enzymes (Hynes, 1977). It is believed that the facB gene product is a positively acting regulatory element necessary for induction of the ACS gene and the genes of the glyoxylate cycle (Hynes and Davis, 1986). In the ascomycetous yeast Yarrowia lipolytica several Acu- mutants were described (Matsuoka et al., 1980, 1984; Barth, 1985) and the structural
194
M. KUJAU, H. WEBER AND G. BARTH GLUCONEOGENESIS
4 4
. 4
PHOSPHOWOLPYRWATE
ASPARTATE
+ +1
FATTY ACIDS cc n-Alkanes
TI0
c
* ETHANOL
OXALOACETATE
CITRATE
PHOSPHOLIPIDS
CYSTEINE
1SERINE
-
SUCCINATE
GLxCINE
mypTopw7 I I THYMIDYLATE
METHYLTETRAHYDRO
FOLATE Figure 1. Scheme of the glyoxylate cycle and related pathways including main enzymes necessary for production of acetylcoenzyme A and function of the glyoxylate shunt. Excluding glyoxylate, all intermediates of this cycle occur also in the tricarboxylic acid cycle, which is not completely shown in this scheme. ACS = acetyl-coenzymeA synthetase, ACO = aconitase, CIS =&rate synthetase, ICL = isocitrate lyase, MAS = malate synthase, MDH = malate dehydrogenase, T I 0 = thiolase.
gene of ICL (ZCL1) was characterized (Barth and Weber, 1987) and cloned (Barth et al., 1990). This fatty acid-assimilating yeast offers the possibility to distinguish easily by growth pattern between ACSless and glyoxylate cycle-defective mutants. The aim of this work was to select mutants which exhibit no or reduced activity of ACS in order to understand the regulation of this pathway in more detail. Among the isolated Acu- mutants, two phenotypical classes (icl2 and dominant Acu- mutants) expressed significantly reduced or no ACS activity. The identification and characterization of the gene ZCL2 as the structural gene of ACS is presented in this paper. Additionally, dominant mutations in a regulatory gene (GPRl = glyoxylate pathway regulation) are described which strongly repress the syntheses of ACS, ICL and MAS on acetate but not on fatty acids as carbon sources. MATERIALS AND METHODS Yeast strains Y. lipolytica strains B204-12C (MATA met6-1 spol-1) and KB50-4 (MA TB leu2-20 spol-1) were
used. Strain B204-12C is an inbred strain derived from strain B201 (Barth and Weber, 1985). Strain KB50-4 arose by a cross of B204- 12C-20 (MA TA leu2-20 metb-1 spol-1) with B204-12D (MATB leu3-4 spol-1) and subsequent backcrosses (five times) to B204-12C. Both strains are characterized by high fertility and the formation of spherical spores. Media and culture conditions Complete medium (YEPD), synthetic minimal medium (MMT) with 2% glucose (MMT-G), 0.5% glycerol (MMT-Gly), 0.4% sodium acetate (MMTA) or 0.5% n-alkanes (chain length from C,, to C,,), and conjugation medium (YM) were prepared as described by Barth and Weber (1983). Diploid strains were induced to sporulate on citrate sporulation medium (CSM) (Barth and Weber, 1985). Amino acids and nucleic bases were supplied at final concentrations of 50 mg/l in MMT. Cultivation of cells took place at 28°C. Acetate-induced cells were prepared according to Barth (1985).
ACETYL-CoA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOLYTICA
195
Preparation of cell-free extracts and enzyme assays
RESULTS
Preparation of cell-free extracts and assay of isocitrate lyase (threo-D-isocitrate glyoxylate lyase, EC4.1.3.1) was done as described by Barth and Weber (1987). Acetyl-coenzyme A synthetase (EC 6.2.1.1) activity was measured by the method of Berg (1956) at pH 7.5 under reducing conditions. Malate synthase (EC 4.1.3.2) was assayed by extinction changes accompanying the cleavage of the S-acyl-bond in acetyl-S-coenzyme A in phosphate buffer at pH 7.0. Protein content was determined by the method of Lowry et al. (1951).
Activity of theglyoxylate cycle in auxotrophic strains
Mutagenesis
We observed large differencesin expression of the glyoxylate cycle enzymes in Y. lipolytica depending on the strain background, which may complicate regulatory studies. Therefore, the two strains B20412C and KB50-4 were constructed as described in Materials and Methods. Both strains show high expression and the same kinetic pattern of the enzymes ACS, ICL and MAS during growth in acetate-containing medium. These strains exhibit high fertility and are suitable for genetic analysis. Cells of Y. lipolytica rapidly increase activities of ACS, ICL and MAS after transfer from MMT-G into MMT-A (Figure 2). Highest activities of these enzymeswere measured at the beginning of the exponential phase of growth. ICL exhibited a maximum of activity between 6 and 8 h of incubation and was reduced after this time to 50-60%, in contrast to the other enzymes. Such kinetics of ICL activity were observed in all strains tested so far, independently of the genetic background. The cause of this phenomenon may be that higher amounts of glyoxylate and succinate are necessary to replenish the cellular pools with precursors for gluconeogenesis and glycine/ serine biosynthesis at the beginning of growth.
Induction of mutations was done by using Nmethyl-N'-nitro-N-nitroso-guanidine. Cells of Y. lipolytica strains B204-12C or KB50-4 were grown in YEPD medium overnight, harvested, washed once and resuspended in 0.9% sodium chloride solution at about 2 x lo7 cells/ml. MNNG was added to a final concentration of 125 pg/ml and the culture was shaken at 28°C. After 40 min cells were harvested, washed, diluted and spread on YEPD agar plates. The frequency of survival was about 1-5%. Acetate-non-utilizing (Acu-) mutants were Selection and characterization of Acu- mutants selected after replica plating of grown colonies To get more insight into the genetic regulation of onto MMT-G and MMT-A agar plates. These Acuthe glyoxylate pathway, 550 Acu- mutants were mutants were further checked by replica plating selected after mutagenesis of strains B204- 12C and onto MMT plates containing 0.5% glycerol, 0 4 % KB5O-4. Four phenotypical classes of mutants ethanol or 0.5% n-alkanes. Selected mutants were could be distinguished by differences in growth on tested for activities of ACS, ICL and MAS. Temperature-sensitivity of ic12 mutants and acetate, ethanol and n-alkanes (instead of fatty revertants of ic12 mutants was tested by incubation acids) (Table 1). Mutants of class I could not grow on all three at 32°C as the restrictive temperature. carbon sources. Complementation analysis has shown that 76 of these mutants belong to the intergenic complementation group ICLI, which is the Complementation and meiotic recombination structural gene of ICL (Barth and Weber, 1987). analysis Figure 3 shows that MAS was fully expressed and Complementation analysis of selected Acu- ACS was about 1.5 times overexpressed in spite of mutants was done by the method described by Barth lack of ICL activity in such mutants. Within class I, and Weber (1984a) using formerly isolated icll and 42 mutants belong to the intergenic complemenic12 mutants (Barth, 1985)as reference strains. Non- tation group ICL3. They express lower activity of complementing Acu- diploid strains did not grow ICL, but have no significant changes in activities of on MMT-A after 7 days. ACS and MAS. The reason for the Acu- phenotype Meiotic recombination analysis to check linkage of ic13 mutants is not known so far. The remaining of different intergenic complementation groups was 191 mutants of class I have no changes in the glyoxycarried out as described by Barth and Weber late cycle and ACS activity. Most probably these (1984b). mutants are blocked in gluconeogenetic steps.
196
M. KUJAU, H. WEBER A N D G. BARTH
200
-
c.
ACS
_ I c I
ICL
----..+ .""
MS
-log(x
100
8 10 cells/ml)
0 c
X 0 0
Y
E
c
2 c a2
0 0
20
10
30
time (h) Figure 2. Specific activities of the enzymes ACS, ICL and MAS in relation to the growth of strain B20412C in MMT-A. Cells were inoculated after 16 h of cultivation in MMT-G at a cell density of 2 x lo7cells/ml in MMT-A.
Table 1. Phenotypical classes and intergenic complementation groups of Acumutants of Y. lipolytica strains B204-12C and KB50-4 which were selected after mutagenesis using N-methyl-N'-nitro-N-nitroso-guanidine. (n.k. =complementation groups are not known) No. of mutants of strain: Class
Phenotype
Complementation group
B204- 12C
KB5M
I
Acu- Eth- Alk-
ICLl ICL3 n.k.
36 31 105 2172
40 11 86 2137
I1
Acu- Eth- Alk+
ICL2 Dominant n.k.
59 7 15 28 1
33 1 28 262
37 23
17 21
111
IV
Acu- Eth+ Alk+ Acu- Eth+ Alk-
n.k. n.k.
Mutants of class I1 grow on n-alkanes but not on acetate or ethanol as carbon source. From these data it could be excluded that these mutants are blocked in the uptake of acetate because ethanol which passively enters the cells was also not degraded. Such mutants are most probably blocked in the activity of ACS. Therefore, fluoroacetate resistance and activities of ACS, ICL and MAS were
~
tested. Only i d 2 mutants are resistant to the toxic agent fluoroacetate, which is a characteristic feature of ACS-defective mutants in A . nidulans (Apirion, 1965; Armitt et al., 1976). These i d 2 mutants show no or a strongly reduced ACS activity (Figure 4). ICL and MAS activities were not detectable or also strongly reduced. But there are differences in enzyme activities of id2 mutants dependent on level
197
ACETYL-CoA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOLYTICA
I5O
-
ICL
50
0 0
20
10
30
tlme (h) Figure 3. Specific activities of the enzymes ACS, ICL and MAS in the icll mutant B20412C-108 during incubation in MMT-A wherein no growth ofcells occurred.
of residual activity of ACS. In strain B204-12C-14 (ic12-14), which cannot grow on acetate or ethanol, no activity of ACS was measured and therefore no induction of ICL or MAS occurred (Figure 4a). Some of the ic12 mutants exhibit a leaky phenotype. In mutant B204- 12C-94 (ic12-94), which grew very slowly on acetate and ethanol, a small increase of ACS activity was observed after prolonged incubation, resulting in slightly enhanced activities of ICL and MAS (Figure 4b). However, ACS activity was not high enough to fully induce the synthesis of the glyoxylate cycle enzymes, not even after prolonged incubation. Besides the ZCL2 complementation group, seven mutants of strain B204-12C and one of strain KB504 were identified which expressed a dominant Acuphenotype. Meiotic recombination analysis has demonstrated that these mutants are unlinked to locus ZCL2. These mutants will be characterized later in this paper in more detail. Mutants ofclass I11 cannot grow on acetate; however, they grow normally on ethanol or n-alkanes as carbon source. These mutants express no activity of ACS, resulting in lack of the glyoxylate cycle during incubation on acetate. However, normal levels of these enzyme activities were measured during growth on ethanol or n-alkanes as carbon source. Therefore, it was assumed that these mutants are most probably blocked in the uptake of acetate. Nothing is known about the background of the phenotype of class IV mutants. No changes of
glyoxylate cycle activity were observed in these mutants. Selection and characterization of temperaturesensitive (ts) mutants of ICL2 It is generally assumed that ts mutants help to identify the structural gene of an enzyme. Therefore, a search was done to select ts id2 mutants and ts revertants of ic12 mutants. All isolated id2 mutants grew at 32°C but one strain (B204-12C-28) was detected to be cold-sensitive (cs). This cs mutant grew slowly at 32°C but very slowly at 24°C and expressed higher ACS activities at 32°C than at 24°C (Table 2). Two revertants of two different ic12 mutants (Tsl of ic12/18 and Ts4 of ic12-14) exhibited a ts phenotype. The two ts revertants express very low activities of ACS at 32°C but higher activities at 24"C, which were sufficient for growth (Table 2). A time-dependent heat-inactivation test of ACS at 35°C was carried out with cell extracts of the wildtype strain B204-12C, the ts revertant of B204-12C18, and the cs mutant B204-12C-28 (Figure 5). The ACS enzymes of the wild type and the cs mutant are stable at 35°C over a long incubation time in contrast to the ACS of the ts revertant, which was quickly inactivated at 35°C. This result shows clearly that the ts phenotype of revertant Tsl is caused by the temperature sensitivity of the enzyme ACS. Meiotic recombination mapping of the ts reversion in strain B204- 12C-18ts and the cs mutation in
198
M. KUJAU. H. WEBER AND G. BARTH
a i
5
-
i 0
---@---
ACS
ICL
MS
20
10
30
t lme (h)
b 40
I
30 ACS ICL
20
MS
10
0 0
20
10
30
tlme (h) Figure 4. Specific activities of the enzymes ACS, ICL and MAS in two id2 mutants which showed no growth (a, strain B204-12C-14) or leaky growth (b, strain B204-12C-94) in MMT-A.
strain B204-12C-28 has shown that both mutations are localized in gene ZCL2. Taken together, the results strongly suggest that the gene ZCL2 encodes the monomer of ACS. Gene dosage efect on ACS activity and its influence on enzymes of the glyoxylate pathway
Heterozygous diploid strains were analysed to detect gene dosage effects, which are often observed for genes which encode monomers of oligomeric enzymes. Heteroallelic ZCL2/icl2 strains expressed
only 50-70% of ACS activity compared with homoallelic ZCL2/ZCL2 strains (Table 3). This is higher than expected for an oligomeric enzyme. However, this reduced ACS activity is sufficient for nearly full expression of ICL (Table 3). ACS activities from 0.5 to 20% were observed in heteroallelic icl2/icl2 diploids and haploid leaky ic12 mutants (Figure 4b). Such a low level was not enough for full induction of ICL and MAS. That means that a minimal level of ACS activity of about 50% is needed for complete induction of ICL synthesis in Y . lipolytica, similar to A . niduluns (Armitt et al., 1976).
I99
ACETYL-COA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWA LIPOL YTICA
Table 2. Growth on acetate and specific activities of ACS and ICL of the two ts revertants Tsl (revertant of icI2 mutant B204- 12C- 18) and Ts4 (revertant of id2 mutant B204- 12C-24) and one cs mutant during incubation at 24°Cand 32°C. Specific activities are expressed as mU/mg protein. (WT means unmutated genes of the glyoxylate cycle) Specific activities after 8 h of incubation in MMT-A at:
Growth on MMT-A after 7 days at: Strain
Related genotype
B204- 12C B204- 12C-18 B204- 12C-24 Ts 1 Ts4 B204- 12C-28
icI2-18 ic12-24 icI2-18ts icI2-24ts ic12-28
WT
24°C 24°C
32°C
+++ -
+++
-
++ ++ -
-
++
32°C
ACS
ICL
ACS
ICL
39.3 0.4 0.2 9.8 6.7 2.4
143.2 19.6 17.3 45.0 58.9 52.6
37.5 0.5 0.2 1.8 2.2 9.7
126.0 24.2 17.5 24.3 36.0 72.8
120 100
8204- l2C-28
80
B204-12C-18ts
60
40
2o
1
o !
0
I
I
I
20
40
60
80
time (min) Figure 5. Heat inactivation of ACS in cell extracts of strain B204-12C (WT), cs mutant B204-12C-28, and ts revertant TsI of zcl2 mutant B204-12C-18 during incubation at 35°C.
In some of the diploid strains, overexpression of triggered by glyoxylate and/or succinate, which are MAS occurred, although there is only about half of formed from isocitrate by ICL. the ACS activity and nearly full expression of ICL activity (Table 3, strain KB85). The reason for this Intragenic complementation analysis of gene ICL2 result is not yet understood. Intragenic complementation is a common feature As in haploid strains (Figure 3), overexpression of oligomeric enzymes and it was demonstrated of ACS of 40-70% was observed in ICLllicll and that it occurs also in genes LYSl and ICLl of Y . icll/icll strains (Table 3). However, this did not lipolytica (Gaillardin and Heslot, 1979; Barth and occur in heterozygous strains which possess only Weber, 1987). Therefore, a complementation analyone intact copy of genes ICLl and ICL2 (Table 3, sis was carried out with 62 ic12 mutations to identify strain KB91). These data suggest a feedback regu- intragenic complementation groups. However, no lation of activity or synthesis of ACS, possibly clear intragenic complementation between ic12
200
M. KUJAU, H. WEBER AND G. BARTH
Table 3. Effect of gene dosage on activities of ACS, ICL and MAS in cells of heterozygous diploid strains which were grown for 16 h in MMT-G, harvested, washed and incubated for 6 h in MMT-A (*enzymeactivitieswere measured after 16 h of cultivation of strain KB81 in MMT-G). 100% relative enzyme activities are 49 mU/mg protein of ACS, 173mU/mg protein for ICL and 135 mU/mg protein for MAS. Relative enzyme activities (%) after 6 h of incubation in MMT-A Relevant genotype
Strain KB8 1 KB8 1* KB83 KB85 KB87 KB88 KB89 KB90 KB9 1 KB94 KB97
ICLl / K L I ICL2IICL2 ICL3/ICL3 gprllgprl
ICL
MAS
100
100
100
6 14 140 93
5 81 87 9 18 27 33 47
1 92 146 6 21 12 82 115
170 7
1 6
81 10
0.5 70 57
ICL2/ic12-31 ic12-I2/ICL2 ic12-I 2/ic12-31 ic12-1OO/ic12-23 icl2-1OO/ic12-31 icll-I08/ICLl icll-108/ICL1 ICL2lic12-31 icll -108/ic11-108 GPRl-1lgprl
30
ACS
0.5
I
-
20
__ige__
ICL MS
10
0
0
20
10
30
tlme (hl Figure 6. Specific activities of ACS, ICL and MAS in the dominant mutant B204-12C-112 (GPRI) during incubation in MMT-A wherein no growth of cells occurred.
mutations was found. We observed leaky growth of some ic12/ic12heteroallelic diploid strains, which is most probably not caused by complementation but by the low activity of ACS present in the haploid
parents used. These data, together with the lack of a clear gene dosage effect, suggest a monomeric state of ACS. However, this hypothesis has to be confirmed by further studies.
ACETYL-CoA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOL YTICA
1 2
940
67-
4c L
20 1
no complementation occurred between these or any other icll, ic12, or ic13 mutants. Meiotic recombination analysis was done to detect whether these dominant mutations are localized in one or in different genes. Diploid strains containing these dominant mutations as well as iclllicll and icl2/ic12 strains sporulate normally on CSM. Arising ascospores germinate normally even though they lack glyoxylate cycle activity. The random spore analysis of several diploid strains containing heterozygous dominant mutations gave no Acu’ recombinants among about 200 selected spores of each strain. This study demonstrates that the dominant mutations are localized in one gqne. Further analysis has shown that there is no linkage of this gene to the genes ICLl, ICL2 and ICL3. In conclusion of these results, we called this new gene GPRI (glyoxylate pathway regulation). DISCUSSION
The glyoxylate cycle is a strongly regulated anaplerotic cycle. The induction of synthesis of the two main enzymes of this pathway, ICL and MAS, is dependent on availability of acetyl-coenzyme during growth on C, compounds or fatty acids. The enzyme 0 ACS is needed for production of acetyl-CoA during growth on acetate. The selection of ACS-defective mutants is complicated by the existence of a second pathway for activation of acetate (Nunn, 1986) in several organisms. This pathway seemsnot to exist in fungi. Besidesthree filamentous fungi, Y. lipolytica is Figure 7. Protein pattern in cell extracts of strain B204-12C the first yeast species from which such mutants are (lane I ) and dominant mutant B204-12C-112 ( G P R I ) (lane 2) available. Using these mutants we have several indiafter 6 h of incubation in MMT-A and purified ICL (lane 3) of cations that ACS is encoded by the gene ICL2, which Y . lipolytica strain B204-12C. was described for the first time by Barth (1985) as a gene which is necessary for synthesis of ICL. Our Characterization of dominant Acu- mutants and data on intragenic suppression of mutation ic12-18 in identijication of the gene GPRl ts revertant Tsl, the intragenic localization of the Eight dominant Acu- mutants exhibited a mutation of the cs ic12mutant B204- 12C-28,the gene growth pattern similar to ic12 mutants. However, dosage effect of the ZCL2 gene, and the fluoroacetate growth was slower on n-alkanes compared with ic12 resistance of ic12 mutants fit the conclusion that the mutants. As in ic12 mutants, strongly reduced activi- gene ICL2 encodes the monomer of ACS. ties of ACS and MAS were measured in MMT-A A feedback regulation of ACS by products of the even after 24 h of incubation (Figure 6). No induc- ICL reaction has not been previously demonstrated. tion of ICL activity was observed; rather, a decrease Our data of overexpression of ACS in icll mutants of the bash level of ICL activity occurred in these suggest such a special mechanism for regulation of dominant mutants grown on acetate as carbon the ACS activity in Y. lipolytica. It is hypothesized source (Figure 6). This lack of ICL activity is caused that the products of the ICL reaction (succinate, by absence of induction of the synthesis of the ICL glyoxylate) or a related compound are included in this control. protein, as is shown in Figure 7. The dominant mutants could not be further Y. lipolytica does not need the glyoxylate cycle for differentiated by complementation studies because sporulation and germination of ascospores, as was
z
20,
202 demonstrated by normal sporulation a n d germination of spores of diploid strains without any activity of this pathway. This contrasts with the observation of high activity of this cycle in seeds of several plants (Vanni et al., 1990) in which the glyoxylate bypass is used for conversion of stored fats into gluconeogenetic compounds and energy. However, germination of spores of Y. lipolytica is induced by externally available glucose and intracellularly stored compounds are probably not used as carbon sources for the outgrowth of spores. Only few regulatory mutants in acetate metabolism are known. I n E. coli, genes iclR and fadR were described (Nunn, 1986). These genes encode repressors for the ace-operon including the genes of ICL, MAS a n d a n acetate kinase, but not the ACS gene. Nothing is known about effects of genes iclR and fadR o n synthesis of ACS in E. coli. Among eukaryotes only two types of regulatory mutants VacB, f a d ) affecting acetate metabolism were selected in A . nidulans (Armitt et al., 1976). Mutants in genes facB and facC (= acuC) have a phenotype similar to ACS-defective mutants and express very low activities of ACS, ICL, MAS and glyconeogenetic enzymes. The trans-dominant mutations in the gene GPRl in Y . lipolytica exhibit similar effects on enzyme pattern as facB a n d facC mutations in A . nidulans. It is shown that the gene GPRl encodes a product which is included in expression of genes of the glyoxylate cycle on acetate as carbon source. It remains to be clarified whether the dominant mutations affect the synthesis or stability of acetylcoenzyme A, which results in lack of induction of ICL a n d MAS syntheses, o r whether the product acts directly on all three genes. Therefore, it will be of interest to clone the genes GPRl and ICL2 of the ascomycetous yeast Y . lipolytica to study this system in more detail. ACKNOWLEDGEMENTS Many thanks are due to Sibylle Briickner and Sylke Forste for technical assistance during this work. REFERENCES Apirion, D. (1965). The two way selection of mutants and revertants in respect of acetate utilization and resistance to fluoroacetate in Aspergillus nidulans. Genet. Res. 6,317-329. Armitt, S., McCullough, W. and Roberts, C. F. (1976). Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans. J. Gen. Microbiol. 92,263-282.
M. KUJAU, H. WEBER AND G. BARTH
Barth, G. (1985). Genetic regulation of isocitrate lyase in the yeast Yarrowia lipolytica. Curr. Genet. 10,l 19-124. Barth, G., Linder, P. and Hinnen, A. (1990). Regulation of the glyoxylate cycle in the yeast Yarrowia lipolytica. Yeast 6, spec. iss., S227. Barth, G. and Weber, H. (1983) Genetic studies on the yeast Saccharomycopsis lipolytica. Inactivation and mutagenesis. Z . Allg. Mikrobiol. 23, 147-157. Barth, G. and Weber, H. (1984a). Improved conditions for mating of the yeast Saccharomycopsis lipolytica. Z . Allg. Mikrobiol. 24,403405. Barth, G. and Weber, H. (1984b) Use of nystatin for random spore selection in the yeast Saccharomycopsis lipolytica. Z . Allg. Mikrobiol. 24, 125-127. Barth, G. and Weber, H. (1985). Improvement of sporulation in the yeast Yarrowia lipolytica. Antonie van Leeuwenhoek 51,167-177. Barth, G. and Weber, H. (1987). Genetic analysis of the gene ICLI of the yeast Yarrowia lipolytica. Yeast 3, 255-262. Berg, P. (1956). Acyl adenylates: An enzymatic mechanism of acetate activation. J. Biol. Chern. 222,991-1013. Casselton, L. A. and Casselton, P. J. (1974). Functional aspects of fluoroacetate resistance in Coprinus with special reference to acetyl-CoA synthetase deficiency. Mol. Gen. Genet. 132,255-264. Connerton, I. F., Fincham, J. R. S., Sandeman, R. A. and Hynes, M. J. (1990). Comparison and cross-species expression of the acetyl-CoA synthetase genes of the ascomycete fungi, Aspergillus nidulans and Neurospora crassa. Mol. Microbiol. 4,45 1460. Flavell, R. B. and Fincham, J. R. S. (1968a). Acetatenonutilizing mutants of Neurospora crassa. I. Mutant isolation, complementation studies and linkage relationships. J. Bacteriol. 95, 10561062. Flavell, R. B. and Fincham, J. R. S. (1968b). Acetatenonutilizing mutants of Neurospora crassa. 11. Biochemical deficiencies and the role of certain enzymes. J . Bacteriol. 95, 1063-1068. Gaillardin, C. and Heslot, H. (1979). Evidence for mutations in the structural gene for homocitrate synthase in Saccharomycopsis lipolytica. Mol. Gen. Genet. 172,185-192. Hynes, M. J. (1977). Induction of acetamidase of Aspergillus nidulans by acetate metabolism. J . Bacteriol. 131,770-775. Hynes, M. J. and Davis, M. A. (1986). The amdS gene of Aspergillus nidulans: control by multiple regulatory signals. Bioassay 5 , 123-128. Kornberg, H. L. (1966). The role and control of the glyoxylate cycle in Escherichia coli. Biochem. J . 99, 1-1 1. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Fohn phenol reagent. J . Biol. Chem. 193,265-275. Matsuoka, M., Himeno, T. and Aiba, S. (1984). Characterization of Saccharomycopsis lipolytica mutants that
ACETYL-COA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOLYTICA
express temperature-sensitive synthesis of isocitrate lyase. J. Bucteriol. 157,899-908. Matsuoka, M., Ueda, Y. and Aiba, S. (1980). Role and control of isocitrate lyase in Cundidu lipolyticu. J. Bucteriol. 144,692497. Nunn, W. D. (1986). A molecular view of fatty acid catabolism in Escherichiu coli. Microbiol. Rev. 50, 1 79- 192. Sandeman, R. A. and Hynes, M. J. (1989). Isolation of the fucA (acetyl-CoenzymeA synthetase) and ucuE(ma1ate
203
synthase) genes of Aspergillus niduluns. Mol. Gen. Genet. 218,87-92. Thomas, G. H., Connerton, I. F. and Fincham, J. R. S. (1988). Molecular cloning, identification and transcriptional analysis of genes involved in acetate utilization in Neurosporu crass. Mol. Microbiol. 2,599406. Vanni, P., Giachetti, E., Pintauti, G . and McFadden, B. A. (1990). Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp. Biochem. Physiol. 95B, 43 1 4 5 8 .
VOL.
8 193-203 (1992)
Characterization of Mutants of the Yeast Yarrowia ZipoZ’tica Defective in Acetyl-Coenzyme A Synthetase MARIAN KUJAU, HERBERT WEBER AND GEROLD BARTH*t Institute for Microbiology and Experimental Therapy, Jena, Beutenbergstr. II,D-6900 Jena, Germany Received 22 June 1991
The expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl-coenzyme A, which is produced by acetyl-coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate-non-utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In ic12 mutants, lack of ACS activity resulted in a non-induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the structural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICLI, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPRI =glyoxylate pathway regulation) was detected in which trans-dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source. KEY WORDS -Glyoxylate
pathway; acetyl-coenzyme A synthetase; isocitrate lyase; dominant mutations; Yarrowia
lipolytica.
INTRODUCTION The glyoxylate pathway has to replenish intermediates (succinate, malate) of the tricarboxylic acid cycle during growth on C, compounds or fatty acids for biosynthesis of gluconeogenetic compounds and amino acids (Kornberg, 1966). Furthermore, biosynthesis of glycine, from which heme and serine are formed, starts from glyoxylate during growth on such substrates (Figure 1). The main enzymes of this pathway are isocitrate lyase (ICL), which converts threo-D-isocitrate into glyoxylate and succinate, and malate synthase (MAS), which synthesizes malate from glyoxylate and acetyle-coenzyme A. However, during growth on glucose, serine is formed from 3-P-glycerate via 3-P-hydroxypyruvate and ophosphorserine, and glycine is synthesized from serine. Oxaloacetate arises by other anaplerotic pathways from phosphoenolpyruvate or pyruvate on glycolytic substrates. Therefore, the syntheses of ICL and MAS are repressed during growth on hexoses. *Present address: Department of Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland. tAddressee for correspondence. 0749%503X/92/030193-11 $05.50 1992 by John Wiley & Sons Ltd
0
Acetyl-coenzyme A, which is formed by acetylcoenzyme A synthetase (ACS) from acetate or by thiolase from fatty acids during P-oxidation, induces the synthesis of ICL and MAS during growth on these carbon sources. Mutants deficient in ACS activity are only known from three filamentous fungi. Such mutants were identified on the basis of fluoroacetate resistance in Aspergillus nidulans (Apirion, 1965) and selected among acetate-nonutilizing mutants (Acu-) of Neurospora crassa (Flavell and Fincham, 1968a,b) and Coprinus cinereus (Casselton and Casselton, 1974). Recently, the ACS-encoding genes of the ascomycetous fungi A . nidulans and N . crassa were cloned and sequenced (Thomas et al., 1988; Sandeman and Hynes, 1989; Connerton et al., 1990). Fluoroacetate-resistant mutants CfacB)of A . nidulans were isolated in which loss-of-function inhibits induction of ACS and of glyoxylate cycle enzymes (Hynes, 1977). It is believed that the facB gene product is a positively acting regulatory element necessary for induction of the ACS gene and the genes of the glyoxylate cycle (Hynes and Davis, 1986). In the ascomycetous yeast Yarrowia lipolytica several Acu- mutants were described (Matsuoka et al., 1980, 1984; Barth, 1985) and the structural
194
M. KUJAU, H. WEBER AND G. BARTH GLUCONEOGENESIS
4 4
. 4
PHOSPHOWOLPYRWATE
ASPARTATE
+ +1
FATTY ACIDS cc n-Alkanes
TI0
c
* ETHANOL
OXALOACETATE
CITRATE
PHOSPHOLIPIDS
CYSTEINE
1SERINE
-
SUCCINATE
GLxCINE
mypTopw7 I I THYMIDYLATE
METHYLTETRAHYDRO
FOLATE Figure 1. Scheme of the glyoxylate cycle and related pathways including main enzymes necessary for production of acetylcoenzyme A and function of the glyoxylate shunt. Excluding glyoxylate, all intermediates of this cycle occur also in the tricarboxylic acid cycle, which is not completely shown in this scheme. ACS = acetyl-coenzymeA synthetase, ACO = aconitase, CIS =&rate synthetase, ICL = isocitrate lyase, MAS = malate synthase, MDH = malate dehydrogenase, T I 0 = thiolase.
gene of ICL (ZCL1) was characterized (Barth and Weber, 1987) and cloned (Barth et al., 1990). This fatty acid-assimilating yeast offers the possibility to distinguish easily by growth pattern between ACSless and glyoxylate cycle-defective mutants. The aim of this work was to select mutants which exhibit no or reduced activity of ACS in order to understand the regulation of this pathway in more detail. Among the isolated Acu- mutants, two phenotypical classes (icl2 and dominant Acu- mutants) expressed significantly reduced or no ACS activity. The identification and characterization of the gene ZCL2 as the structural gene of ACS is presented in this paper. Additionally, dominant mutations in a regulatory gene (GPRl = glyoxylate pathway regulation) are described which strongly repress the syntheses of ACS, ICL and MAS on acetate but not on fatty acids as carbon sources. MATERIALS AND METHODS Yeast strains Y. lipolytica strains B204-12C (MATA met6-1 spol-1) and KB50-4 (MA TB leu2-20 spol-1) were
used. Strain B204-12C is an inbred strain derived from strain B201 (Barth and Weber, 1985). Strain KB50-4 arose by a cross of B204- 12C-20 (MA TA leu2-20 metb-1 spol-1) with B204-12D (MATB leu3-4 spol-1) and subsequent backcrosses (five times) to B204-12C. Both strains are characterized by high fertility and the formation of spherical spores. Media and culture conditions Complete medium (YEPD), synthetic minimal medium (MMT) with 2% glucose (MMT-G), 0.5% glycerol (MMT-Gly), 0.4% sodium acetate (MMTA) or 0.5% n-alkanes (chain length from C,, to C,,), and conjugation medium (YM) were prepared as described by Barth and Weber (1983). Diploid strains were induced to sporulate on citrate sporulation medium (CSM) (Barth and Weber, 1985). Amino acids and nucleic bases were supplied at final concentrations of 50 mg/l in MMT. Cultivation of cells took place at 28°C. Acetate-induced cells were prepared according to Barth (1985).
ACETYL-CoA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOLYTICA
195
Preparation of cell-free extracts and enzyme assays
RESULTS
Preparation of cell-free extracts and assay of isocitrate lyase (threo-D-isocitrate glyoxylate lyase, EC4.1.3.1) was done as described by Barth and Weber (1987). Acetyl-coenzyme A synthetase (EC 6.2.1.1) activity was measured by the method of Berg (1956) at pH 7.5 under reducing conditions. Malate synthase (EC 4.1.3.2) was assayed by extinction changes accompanying the cleavage of the S-acyl-bond in acetyl-S-coenzyme A in phosphate buffer at pH 7.0. Protein content was determined by the method of Lowry et al. (1951).
Activity of theglyoxylate cycle in auxotrophic strains
Mutagenesis
We observed large differencesin expression of the glyoxylate cycle enzymes in Y. lipolytica depending on the strain background, which may complicate regulatory studies. Therefore, the two strains B20412C and KB50-4 were constructed as described in Materials and Methods. Both strains show high expression and the same kinetic pattern of the enzymes ACS, ICL and MAS during growth in acetate-containing medium. These strains exhibit high fertility and are suitable for genetic analysis. Cells of Y. lipolytica rapidly increase activities of ACS, ICL and MAS after transfer from MMT-G into MMT-A (Figure 2). Highest activities of these enzymeswere measured at the beginning of the exponential phase of growth. ICL exhibited a maximum of activity between 6 and 8 h of incubation and was reduced after this time to 50-60%, in contrast to the other enzymes. Such kinetics of ICL activity were observed in all strains tested so far, independently of the genetic background. The cause of this phenomenon may be that higher amounts of glyoxylate and succinate are necessary to replenish the cellular pools with precursors for gluconeogenesis and glycine/ serine biosynthesis at the beginning of growth.
Induction of mutations was done by using Nmethyl-N'-nitro-N-nitroso-guanidine. Cells of Y. lipolytica strains B204-12C or KB50-4 were grown in YEPD medium overnight, harvested, washed once and resuspended in 0.9% sodium chloride solution at about 2 x lo7 cells/ml. MNNG was added to a final concentration of 125 pg/ml and the culture was shaken at 28°C. After 40 min cells were harvested, washed, diluted and spread on YEPD agar plates. The frequency of survival was about 1-5%. Acetate-non-utilizing (Acu-) mutants were Selection and characterization of Acu- mutants selected after replica plating of grown colonies To get more insight into the genetic regulation of onto MMT-G and MMT-A agar plates. These Acuthe glyoxylate pathway, 550 Acu- mutants were mutants were further checked by replica plating selected after mutagenesis of strains B204- 12C and onto MMT plates containing 0.5% glycerol, 0 4 % KB5O-4. Four phenotypical classes of mutants ethanol or 0.5% n-alkanes. Selected mutants were could be distinguished by differences in growth on tested for activities of ACS, ICL and MAS. Temperature-sensitivity of ic12 mutants and acetate, ethanol and n-alkanes (instead of fatty revertants of ic12 mutants was tested by incubation acids) (Table 1). Mutants of class I could not grow on all three at 32°C as the restrictive temperature. carbon sources. Complementation analysis has shown that 76 of these mutants belong to the intergenic complementation group ICLI, which is the Complementation and meiotic recombination structural gene of ICL (Barth and Weber, 1987). analysis Figure 3 shows that MAS was fully expressed and Complementation analysis of selected Acu- ACS was about 1.5 times overexpressed in spite of mutants was done by the method described by Barth lack of ICL activity in such mutants. Within class I, and Weber (1984a) using formerly isolated icll and 42 mutants belong to the intergenic complemenic12 mutants (Barth, 1985)as reference strains. Non- tation group ICL3. They express lower activity of complementing Acu- diploid strains did not grow ICL, but have no significant changes in activities of on MMT-A after 7 days. ACS and MAS. The reason for the Acu- phenotype Meiotic recombination analysis to check linkage of ic13 mutants is not known so far. The remaining of different intergenic complementation groups was 191 mutants of class I have no changes in the glyoxycarried out as described by Barth and Weber late cycle and ACS activity. Most probably these (1984b). mutants are blocked in gluconeogenetic steps.
196
M. KUJAU, H. WEBER A N D G. BARTH
200
-
c.
ACS
_ I c I
ICL
----..+ .""
MS
-log(x
100
8 10 cells/ml)
0 c
X 0 0
Y
E
c
2 c a2
0 0
20
10
30
time (h) Figure 2. Specific activities of the enzymes ACS, ICL and MAS in relation to the growth of strain B20412C in MMT-A. Cells were inoculated after 16 h of cultivation in MMT-G at a cell density of 2 x lo7cells/ml in MMT-A.
Table 1. Phenotypical classes and intergenic complementation groups of Acumutants of Y. lipolytica strains B204-12C and KB50-4 which were selected after mutagenesis using N-methyl-N'-nitro-N-nitroso-guanidine. (n.k. =complementation groups are not known) No. of mutants of strain: Class
Phenotype
Complementation group
B204- 12C
KB5M
I
Acu- Eth- Alk-
ICLl ICL3 n.k.
36 31 105 2172
40 11 86 2137
I1
Acu- Eth- Alk+
ICL2 Dominant n.k.
59 7 15 28 1
33 1 28 262
37 23
17 21
111
IV
Acu- Eth+ Alk+ Acu- Eth+ Alk-
n.k. n.k.
Mutants of class I1 grow on n-alkanes but not on acetate or ethanol as carbon source. From these data it could be excluded that these mutants are blocked in the uptake of acetate because ethanol which passively enters the cells was also not degraded. Such mutants are most probably blocked in the activity of ACS. Therefore, fluoroacetate resistance and activities of ACS, ICL and MAS were
~
tested. Only i d 2 mutants are resistant to the toxic agent fluoroacetate, which is a characteristic feature of ACS-defective mutants in A . nidulans (Apirion, 1965; Armitt et al., 1976). These i d 2 mutants show no or a strongly reduced ACS activity (Figure 4). ICL and MAS activities were not detectable or also strongly reduced. But there are differences in enzyme activities of id2 mutants dependent on level
197
ACETYL-CoA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOLYTICA
I5O
-
ICL
50
0 0
20
10
30
tlme (h) Figure 3. Specific activities of the enzymes ACS, ICL and MAS in the icll mutant B20412C-108 during incubation in MMT-A wherein no growth ofcells occurred.
of residual activity of ACS. In strain B204-12C-14 (ic12-14), which cannot grow on acetate or ethanol, no activity of ACS was measured and therefore no induction of ICL or MAS occurred (Figure 4a). Some of the ic12 mutants exhibit a leaky phenotype. In mutant B204- 12C-94 (ic12-94), which grew very slowly on acetate and ethanol, a small increase of ACS activity was observed after prolonged incubation, resulting in slightly enhanced activities of ICL and MAS (Figure 4b). However, ACS activity was not high enough to fully induce the synthesis of the glyoxylate cycle enzymes, not even after prolonged incubation. Besides the ZCL2 complementation group, seven mutants of strain B204-12C and one of strain KB504 were identified which expressed a dominant Acuphenotype. Meiotic recombination analysis has demonstrated that these mutants are unlinked to locus ZCL2. These mutants will be characterized later in this paper in more detail. Mutants ofclass I11 cannot grow on acetate; however, they grow normally on ethanol or n-alkanes as carbon source. These mutants express no activity of ACS, resulting in lack of the glyoxylate cycle during incubation on acetate. However, normal levels of these enzyme activities were measured during growth on ethanol or n-alkanes as carbon source. Therefore, it was assumed that these mutants are most probably blocked in the uptake of acetate. Nothing is known about the background of the phenotype of class IV mutants. No changes of
glyoxylate cycle activity were observed in these mutants. Selection and characterization of temperaturesensitive (ts) mutants of ICL2 It is generally assumed that ts mutants help to identify the structural gene of an enzyme. Therefore, a search was done to select ts id2 mutants and ts revertants of ic12 mutants. All isolated id2 mutants grew at 32°C but one strain (B204-12C-28) was detected to be cold-sensitive (cs). This cs mutant grew slowly at 32°C but very slowly at 24°C and expressed higher ACS activities at 32°C than at 24°C (Table 2). Two revertants of two different ic12 mutants (Tsl of ic12/18 and Ts4 of ic12-14) exhibited a ts phenotype. The two ts revertants express very low activities of ACS at 32°C but higher activities at 24"C, which were sufficient for growth (Table 2). A time-dependent heat-inactivation test of ACS at 35°C was carried out with cell extracts of the wildtype strain B204-12C, the ts revertant of B204-12C18, and the cs mutant B204-12C-28 (Figure 5). The ACS enzymes of the wild type and the cs mutant are stable at 35°C over a long incubation time in contrast to the ACS of the ts revertant, which was quickly inactivated at 35°C. This result shows clearly that the ts phenotype of revertant Tsl is caused by the temperature sensitivity of the enzyme ACS. Meiotic recombination mapping of the ts reversion in strain B204- 12C-18ts and the cs mutation in
198
M. KUJAU. H. WEBER AND G. BARTH
a i
5
-
i 0
---@---
ACS
ICL
MS
20
10
30
t lme (h)
b 40
I
30 ACS ICL
20
MS
10
0 0
20
10
30
tlme (h) Figure 4. Specific activities of the enzymes ACS, ICL and MAS in two id2 mutants which showed no growth (a, strain B204-12C-14) or leaky growth (b, strain B204-12C-94) in MMT-A.
strain B204-12C-28 has shown that both mutations are localized in gene ZCL2. Taken together, the results strongly suggest that the gene ZCL2 encodes the monomer of ACS. Gene dosage efect on ACS activity and its influence on enzymes of the glyoxylate pathway
Heterozygous diploid strains were analysed to detect gene dosage effects, which are often observed for genes which encode monomers of oligomeric enzymes. Heteroallelic ZCL2/icl2 strains expressed
only 50-70% of ACS activity compared with homoallelic ZCL2/ZCL2 strains (Table 3). This is higher than expected for an oligomeric enzyme. However, this reduced ACS activity is sufficient for nearly full expression of ICL (Table 3). ACS activities from 0.5 to 20% were observed in heteroallelic icl2/icl2 diploids and haploid leaky ic12 mutants (Figure 4b). Such a low level was not enough for full induction of ICL and MAS. That means that a minimal level of ACS activity of about 50% is needed for complete induction of ICL synthesis in Y . lipolytica, similar to A . niduluns (Armitt et al., 1976).
I99
ACETYL-COA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWA LIPOL YTICA
Table 2. Growth on acetate and specific activities of ACS and ICL of the two ts revertants Tsl (revertant of icI2 mutant B204- 12C- 18) and Ts4 (revertant of id2 mutant B204- 12C-24) and one cs mutant during incubation at 24°Cand 32°C. Specific activities are expressed as mU/mg protein. (WT means unmutated genes of the glyoxylate cycle) Specific activities after 8 h of incubation in MMT-A at:
Growth on MMT-A after 7 days at: Strain
Related genotype
B204- 12C B204- 12C-18 B204- 12C-24 Ts 1 Ts4 B204- 12C-28
icI2-18 ic12-24 icI2-18ts icI2-24ts ic12-28
WT
24°C 24°C
32°C
+++ -
+++
-
++ ++ -
-
++
32°C
ACS
ICL
ACS
ICL
39.3 0.4 0.2 9.8 6.7 2.4
143.2 19.6 17.3 45.0 58.9 52.6
37.5 0.5 0.2 1.8 2.2 9.7
126.0 24.2 17.5 24.3 36.0 72.8
120 100
8204- l2C-28
80
B204-12C-18ts
60
40
2o
1
o !
0
I
I
I
20
40
60
80
time (min) Figure 5. Heat inactivation of ACS in cell extracts of strain B204-12C (WT), cs mutant B204-12C-28, and ts revertant TsI of zcl2 mutant B204-12C-18 during incubation at 35°C.
In some of the diploid strains, overexpression of triggered by glyoxylate and/or succinate, which are MAS occurred, although there is only about half of formed from isocitrate by ICL. the ACS activity and nearly full expression of ICL activity (Table 3, strain KB85). The reason for this Intragenic complementation analysis of gene ICL2 result is not yet understood. Intragenic complementation is a common feature As in haploid strains (Figure 3), overexpression of oligomeric enzymes and it was demonstrated of ACS of 40-70% was observed in ICLllicll and that it occurs also in genes LYSl and ICLl of Y . icll/icll strains (Table 3). However, this did not lipolytica (Gaillardin and Heslot, 1979; Barth and occur in heterozygous strains which possess only Weber, 1987). Therefore, a complementation analyone intact copy of genes ICLl and ICL2 (Table 3, sis was carried out with 62 ic12 mutations to identify strain KB91). These data suggest a feedback regu- intragenic complementation groups. However, no lation of activity or synthesis of ACS, possibly clear intragenic complementation between ic12
200
M. KUJAU, H. WEBER AND G. BARTH
Table 3. Effect of gene dosage on activities of ACS, ICL and MAS in cells of heterozygous diploid strains which were grown for 16 h in MMT-G, harvested, washed and incubated for 6 h in MMT-A (*enzymeactivitieswere measured after 16 h of cultivation of strain KB81 in MMT-G). 100% relative enzyme activities are 49 mU/mg protein of ACS, 173mU/mg protein for ICL and 135 mU/mg protein for MAS. Relative enzyme activities (%) after 6 h of incubation in MMT-A Relevant genotype
Strain KB8 1 KB8 1* KB83 KB85 KB87 KB88 KB89 KB90 KB9 1 KB94 KB97
ICLl / K L I ICL2IICL2 ICL3/ICL3 gprllgprl
ICL
MAS
100
100
100
6 14 140 93
5 81 87 9 18 27 33 47
1 92 146 6 21 12 82 115
170 7
1 6
81 10
0.5 70 57
ICL2/ic12-31 ic12-I2/ICL2 ic12-I 2/ic12-31 ic12-1OO/ic12-23 icl2-1OO/ic12-31 icll-I08/ICLl icll-108/ICL1 ICL2lic12-31 icll -108/ic11-108 GPRl-1lgprl
30
ACS
0.5
I
-
20
__ige__
ICL MS
10
0
0
20
10
30
tlme (hl Figure 6. Specific activities of ACS, ICL and MAS in the dominant mutant B204-12C-112 (GPRI) during incubation in MMT-A wherein no growth of cells occurred.
mutations was found. We observed leaky growth of some ic12/ic12heteroallelic diploid strains, which is most probably not caused by complementation but by the low activity of ACS present in the haploid
parents used. These data, together with the lack of a clear gene dosage effect, suggest a monomeric state of ACS. However, this hypothesis has to be confirmed by further studies.
ACETYL-CoA SYNTHETASE-DEFECTIVE MUTANTS OF YARROWIA LIPOL YTICA
1 2
940
67-
4c L
20 1
no complementation occurred between these or any other icll, ic12, or ic13 mutants. Meiotic recombination analysis was done to detect whether these dominant mutations are localized in one or in different genes. Diploid strains containing these dominant mutations as well as iclllicll and icl2/ic12 strains sporulate normally on CSM. Arising ascospores germinate normally even though they lack glyoxylate cycle activity. The random spore analysis of several diploid strains containing heterozygous dominant mutations gave no Acu’ recombinants among about 200 selected spores of each strain. This study demonstrates that the dominant mutations are localized in one gqne. Further analysis has shown that there is no linkage of this gene to the genes ICLl, ICL2 and ICL3. In conclusion of these results, we called this new gene GPRI (glyoxylate pathway regulation). DISCUSSION
The glyoxylate cycle is a strongly regulated anaplerotic cycle. The induction of synthesis of the two main enzymes of this pathway, ICL and MAS, is dependent on availability of acetyl-coenzyme during growth on C, compounds or fatty acids. The enzyme 0 ACS is needed for production of acetyl-CoA during growth on acetate. The selection of ACS-defective mutants is complicated by the existence of a second pathway for activation of acetate (Nunn, 1986) in several organisms. This pathway seemsnot to exist in fungi. Besidesthree filamentous fungi, Y. lipolytica is Figure 7. Protein pattern in cell extracts of strain B204-12C the first yeast species from which such mutants are (lane I ) and dominant mutant B204-12C-112 ( G P R I ) (lane 2) available. Using these mutants we have several indiafter 6 h of incubation in MMT-A and purified ICL (lane 3) of cations that ACS is encoded by the gene ICL2, which Y . lipolytica strain B204-12C. was described for the first time by Barth (1985) as a gene which is necessary for synthesis of ICL. Our Characterization of dominant Acu- mutants and data on intragenic suppression of mutation ic12-18 in identijication of the gene GPRl ts revertant Tsl, the intragenic localization of the Eight dominant Acu- mutants exhibited a mutation of the cs ic12mutant B204- 12C-28,the gene growth pattern similar to ic12 mutants. However, dosage effect of the ZCL2 gene, and the fluoroacetate growth was slower on n-alkanes compared with ic12 resistance of ic12 mutants fit the conclusion that the mutants. As in ic12 mutants, strongly reduced activi- gene ICL2 encodes the monomer of ACS. ties of ACS and MAS were measured in MMT-A A feedback regulation of ACS by products of the even after 24 h of incubation (Figure 6). No induc- ICL reaction has not been previously demonstrated. tion of ICL activity was observed; rather, a decrease Our data of overexpression of ACS in icll mutants of the bash level of ICL activity occurred in these suggest such a special mechanism for regulation of dominant mutants grown on acetate as carbon the ACS activity in Y. lipolytica. It is hypothesized source (Figure 6). This lack of ICL activity is caused that the products of the ICL reaction (succinate, by absence of induction of the synthesis of the ICL glyoxylate) or a related compound are included in this control. protein, as is shown in Figure 7. The dominant mutants could not be further Y. lipolytica does not need the glyoxylate cycle for differentiated by complementation studies because sporulation and germination of ascospores, as was
z
20,
202 demonstrated by normal sporulation a n d germination of spores of diploid strains without any activity of this pathway. This contrasts with the observation of high activity of this cycle in seeds of several plants (Vanni et al., 1990) in which the glyoxylate bypass is used for conversion of stored fats into gluconeogenetic compounds and energy. However, germination of spores of Y. lipolytica is induced by externally available glucose and intracellularly stored compounds are probably not used as carbon sources for the outgrowth of spores. Only few regulatory mutants in acetate metabolism are known. I n E. coli, genes iclR and fadR were described (Nunn, 1986). These genes encode repressors for the ace-operon including the genes of ICL, MAS a n d a n acetate kinase, but not the ACS gene. Nothing is known about effects of genes iclR and fadR o n synthesis of ACS in E. coli. Among eukaryotes only two types of regulatory mutants VacB, f a d ) affecting acetate metabolism were selected in A . nidulans (Armitt et al., 1976). Mutants in genes facB and facC (= acuC) have a phenotype similar to ACS-defective mutants and express very low activities of ACS, ICL, MAS and glyconeogenetic enzymes. The trans-dominant mutations in the gene GPRl in Y . lipolytica exhibit similar effects on enzyme pattern as facB a n d facC mutations in A . nidulans. It is shown that the gene GPRl encodes a product which is included in expression of genes of the glyoxylate cycle on acetate as carbon source. It remains to be clarified whether the dominant mutations affect the synthesis or stability of acetylcoenzyme A, which results in lack of induction of ICL a n d MAS syntheses, o r whether the product acts directly on all three genes. Therefore, it will be of interest to clone the genes GPRl and ICL2 of the ascomycetous yeast Y . lipolytica to study this system in more detail. ACKNOWLEDGEMENTS Many thanks are due to Sibylle Briickner and Sylke Forste for technical assistance during this work. REFERENCES Apirion, D. (1965). The two way selection of mutants and revertants in respect of acetate utilization and resistance to fluoroacetate in Aspergillus nidulans. Genet. Res. 6,317-329. Armitt, S., McCullough, W. and Roberts, C. F. (1976). Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans. J. Gen. Microbiol. 92,263-282.
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