T H E JOURNAL OF BIOLOGICAL CHEMISTRY vol. 264. No. 19, Issue of July 5, pp. 11204-11210.1989 Printed in U. S. A.

Metabolic Studies on Citrate Synthase Mutants of Yeast A CHANGE IN PHENOTYPE FOLLOWING TRANSFORMATION WITH AN INACTIVE ENZYME*

(Received for publication, December 16, 1988)

Gyula KispalS, Claudia T. Evans, Craig Malloyl, and Paul A. Srerell From the Pre-Clinical Science Unit and §Cardiology Unit of the Veterans Administration Medical Center and Department of Biochemistry of The University of Texas Southwestern Medical Center, Dallos, Texas 75216

We have studied the growth on acetate, the metabo- lism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous ob- servation that cytosolic citrate synthase is not neces- sary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and a-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagenesis, we constructed two separate CS1 pro- teins with mutations in the enzyme’s active site. The mitochondria of cells carrying either site-directed mu- tagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was re- placed with a glycine (CSl/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CSl/D414G), no growth on acetate or changes in other parameters was observed. We propose that the char- acteristics of the strain carrying the CS 1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally un- changed H313G protein.

There is little doubt that organization of sequential en- zymes of many metabolic sequences occurs in most cells. These organized sequences cover the range of stability from multifunctional proteins which are covalently linked sequen- tial active sites to easily dissociable or dynamic associations of sequential enzymes (1). For the Krebs tricarboxylic acid cycle, many lines of evidence indicate that an organized system exists within the mitochondrial matrix. This evidence includes in vitro experiments with purified enzymes that showed specific interactions between sequential Krebs tricar- boxylic acid cycle enzymes (see Ref. 2 for review). We recently showed that gentle disruption of mitochondria yields a prep- aration of bound Krebs tricarboxylic acid cycle enzymes,

* This research was supported by grants from the Veterans Admin- istration, National Institutes of Health, and the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: The Institute of Biochemistry, University Med- ical School, H-7624 Pecs, Szigetu u t 12, Hungary.

1 To whom reprint requests should be addressed: VA Medical Center, Research Service (151B), 4500 S. Lancaster Rd., Dallas, TX 75216. Tel.: (214) 376-5451; Ext. 5604.

termed a metabolon, which exhibits a kinetic advantage in several coupled enzyme reactions over a soluble mixture of the same enzyme reactions (3). In addition, a recent report showed that five enzymes of the Krebs tricarboxylic acid cycle of Escherichia coli could be isolated as a high molecular weight complex (4).

Although the in vitro evidence for such cellular organization is good, additional data concerning the existence and func- tional importance of these complexes i n vivo are desirable. One approach is to derive an enzyme of a complex that is fully active enzymatically but altered so as to have lost its ability to bind to its sequential partners. The metabolic be- havior of cells containing such an altered enzyme could then be compared with cells containing the wild-type enzyme. Unfortunately, there is no known system for which binding sites that participate in loose multienzyme complexes have been identified. Another strategy is to compare the metabo- lism of cells which lack a certain enzyme activity using ones in which the whole protein is missing and ones in which the protein is present but inactive. In the first case, the multien- zyme complex in which the enzyme operates would be incom- plete because of the missing protein. In the latter case, the complex would be physically complete since the whole protein and its binding sites are present, but activity would be missing by a change in an essential amino acid residue in the active site of the enzyme. If a metabolic difference is detected between the two cell types, one logical explanation could be a difference in the catalytic efficiency of the complex containing an intact protein without an enzyme activity compared with that of a disrupted complex which lacks the protein as well as the activity.

We have chosen to examine this hypothesis in yeast and in the pathways which utilize the citrate synthase enzymes for several reasons. Yeast cells, unlike most eukaryotic cells, contain both a cytosolic (CS2)’ and mitochondrial (CSl) citrate synthase, the former used in the glyoxylate cycle and the latter in the Krebs tricarboxylic acid cycle (5). One might imagine that the lack of either the mitochondrial or cytosolic enzyme would be partially compensated for by the activity of the remaining enzyme, but our earlier results (6) showed that CS2 cannot replace CS1 activity. In addition, other studies in our laboratory on site-directed mutagenesis of pig citrate synthase have identified several residues in citrate synthase needed for citrate synthase activity.’ We also have available

The abbreviations used are: CS2, cytosolic citrate synthase; CS1, mitochondrial citrate synthase; MES, 4-morpholineethanesulfonic acid; the convention for site-directed mutagenesis uses the one-letter amino acid code with the original residue and its position number followed by the newly inserted residue. Thus, H313G indicates that the original histidine at position 313 of CS1 has been replaced with a glycine.

-

* C. T. Evans and P. A. Srere, unpublished data.

11204

Metabolism of Yeast Citrate Synthase Mutants 11205

yeast mutants lacking the citrate synthases and have previ- ously studied their metabolic behavior (6). In this study, we compared the growth and metabolic behavior of strains car- rying one of three mitochondrial citrate synthase mutations: one mutant lacked the citrate synthase molecule, and two others derived by site-directed mutagenesis contained inactive but structurally intact citrate synthase molecules.

MATERIALS AND METHODS

Strains-The parental strain PSY142 (Mata, ura3-52,ku2-2,leu2- 112, lys2-801) was used to make derivatives CS1-, CS2-, and CSl-CS2- identical with those in BWG1-7a which has been described previously (7). In the CS1- mutant, the CITl gene was disrupted with LEU2 (CITl::LEU2) and in the CS2- mutant, CIT2 was disrupted with URA3 (CZT2::URA3). These were obtained from Dr. L. Guar- ente. The strains are described in Table I.

Media-Complete medium contained 2% Difco peptone, 1% yeast extract (Difco), and 35 pg/ml ampicillin. Two % glucose or galactose were used unless otherwise noted. Minimal medium contained 0.7% yeast nitrogen base (Difco), ampicillin (35 pglml), L-lysine (30 pg/ ml), L-leucine (30 pg/ml), uracil (10 pg/ml) (8), and the nutritional factors required for yeast (8). Acetate was added to 0.5% along with 50 mM MES, pH 5.5. Growth rate was determined by measuring the turbidity of cultures at 600 nm.

Determination of Enzyme Activities-Cells were grown to mid- growth phase, chilled, and harvested by centrifugation at 4 "C. Cells were resuspended in 50 mM Tris-HC1 buffer, pH 7.4, containing 1 mM EDTA, 1 mM benzamidine HCl (buffer A) and disrupted with glass beads. The suspension was centrifuged at 20,000 X g for 15 min at 4 "C, and the clear supernatant solution was used for determination of enzyme activities. For measurement of mitochondrial enzyme activities, the mitochondria were disrupted in buffer A containing 1% Triton X-100.

The following enzymes were assayed according to standard proce- dures: citrate synthase (9), aconitase (lo), malate dehydrogenase ( l l ) , fumarase (12), succinate dehydrogenase (13), pyruvate dehydrogenase complex and a-ketoglutarate dehydrogenase complex (14), NADH- cytochrome c oxidoreductase and cytochrome c oxidase (15), isocitrate lyase and malate synthase (16), fructose-1,6-bisphosphatase (17), phosphoenolpyruvate carboxykinase (18), and glutamate dehydrogen- ase and alcohol dehydrogenase (19). Mitochondria were prepared following the procedure of Daum et al. (20). Mitochondrial respiration was measured using a Clark oxygen electrode with substrate concen- trations as described (21). Protein was determined by the method of Lowry et ai. (22).

Utilization of [2-'3C]Acetate-Cells were grown in complete me- dium with galactose to mid-growth phase and harvested by centrifu- gation at room temperature. Cells were resuspended in 20 ml of incubation medium (0.7% yeast nitrogen base, nutritional factors, 0.5% Na[2-13C]acetate, 50 mM MES, pH 5.5, at 5 mg/ml protein concentration) and incubated for 20 and 40 min. The reaction was stopped by the addition of perchloric acid to 0.5 M final concentration.

TABLE I Plasmids and S. cerevisiae strains that contain the DNAs encoding

the mutant and non-mutant yeast CS proteins Plasmid or strain Relevant eenotvue or Dhenotwe Source or ref.

Plasmid

pFCSl YEp352 YEp352/CS1 YEp352/CSl/ D414G YEp352/CSl/ H313G pKlO pKCS2

PFL-1

Yeast PSY142

cs1- cs2-

Ap' Tc' 2~ URA3

LACZ' URA3 2p Ap' YEp352/CITl YEp352/CITl with Asp414

substituted with Gly YEp352/CITl with His313

URA3 GAL UAS 2p Ap' substituted with Gly

pKlOICIT2 (YCS2/pK10)

pFL-l/CITl (YCSl/pFL-l) (24) (24)

This study This study This study

This study

G. Schatz This study

Mat& urd-52 ku2-2 ku2-112 (6)

Psy142/CITl::LEU2 Psv142/CIT2::URA3

L. Guarente L. Guarente

1 ~ ~ 2 - 8 0 1

The neutralized perchloric acid extracts were used for NMR analysis. NMR spectra were obtained in a Nicolet NT300 spectrometer with

Bruker probes with the samples spinning at 20 Hz. Field frequency lock was used (D20 or CDCI,). Carbon spectra were obtained in the 10-mm probe under proton broad-band decoupling using a multiple pulse and phase-shifted decoupling sequence (23). All carbon-13 spectra were collected with a 45" carbon pulse utilizing 8,192 points over a 16,000 Hz spectral width. Approximately 5,000 scans were obtained for the aqueous solutions at a repetition time of 6.5 s. Prior to Fourier transformation, the spectra were zero-filled (to improve digital resolution) and in some cases multiplied by an exponential function of 1-3 Hz (to improve signal-to-noise). Peaks were identified according to their location, and the heights were compared with the acetate peak to determine the relative incorporation 13C label into each peak.

Recombinant Techniques-CS1 gene cloned in vector pFL-1 (pFCS1) was a kind gift of G. Schatz (24). A KpnI-PstI fragment containing CS1 was also cloned into vector YEp352 (YCSl/YEp352) at compatible sites. An EcoRI-EcoRI fragment containing the whole CS2 gene was cloned into the multicopy vector pKlO (pKCS2). All recombinant DNA manipulations were done according to Maniatis et al. (25). Mutagenesis of the CS1 gene was performed with the MUTA- GENE@ in vitro mutagenesis kit (Bio-Rad). The KpnI-PstI fragment containing the CS1 gene was cloned into M13mp18 vector and His- 313 (CSl/H313G) and Asp-414 (CSl/D414G) were individually re- placed with Gly (Table I). The mutations were verified by [36S]dideo~y nucleotide sequencing (26). The mutated genes were subcloned into the YEp352 vector and used to transform PSY142 CS1- yeast cells (27). The transformed cells were plated on complete medium with glucose. Individual transformed colonies were purified by restreaking 10 times on complete medium with glucose. Ten colonies from each cell line were selected at each passage, suspended in buffer, disrupted with glass beads, and assayed for citrate synthase activity (as above) and for CS1 protein by the Western blot method. The colonies retained for further studies showed CS1 protein but had no citrate synthase activity. These cells showed no citrate synthase activity even after approximately 100 generations.

Procedures for sodium dodecyl sulfate-polyacrylamide electropho- resis and transfer of proteins from sodium dodecyl sulfate-polyacryl- amide gels to nitrocellulose membrane and the processing of nitro- cellulose blots have been described previously (28). The anti-yeast citrate synthase serum was prepared according to the procedure of Alam et al. (29) using purified yeast CS1 enzyme. The second antibody was alkaline phosphatase conjugated anti-rabbit IgG (Sigma). The immunoreaction was developed by BCIP/NBT phosphatase reaction system (Kirkegard and Perry Labs).

RESULTS

Growth of Citrate Synthase Mutants on Various Carbon Sources-We have examined the growth of citrate synthase mutants on acetate and on acetate with added glucose or glutamate (Table 11). Growth of the parental PSY142 strain and of the CS2- (disruption of the cytosolic enzyme) occurred on acetate alone and was unaffected by the addition of glucose or glutamate. The CS1- strain containing the pKlO vector (CSl-+pKlO) did not grow on acetate alone or acetate sup- plemented with glutamate. When glucose was added to acetate a small amount of growth was observed but this was probably growth on the glucose.

TABLE I1 Growth of PSY142 and its transformants on minimal

media with different carbon sources Cells were grown on minimal media plates supplemented with the

appropriate nutritional requirements as indicated in the methods. (- is no growth, + is slight griwth, and ++ is good growth). .. ~

1% 0.5% 0.5% acetatel 0.5% acetate/ acetate acetate 0.05% glucose 0.05% glutamate

Parental cs2-

++ ++ ++ ++ ++ ++ ++ ++

CSl-/pKlO - - + - CSl-/pKCS2 - CSl-/pFCSl ++ ++

+ ++ ++

- -

11206 Metabolism of Yeast Citrate Synthase Mutants

To see if an increase in the quantity of CS2 in the CS1- cell would allow the cells to grow on acetate, a CS1- strain containing YCS2IpK10 (CSl-+YCSB/pKlO) was constructed which, when grown on galactose, had an eight time greater amount of CS2 than the CS1- cell (see below). This result confirmed the high copy number nature of pK10. These cells did not grow on acetate or acetate plus glutamate. When CS1 was restored to the cell (CSl-+YCSl/pFL-l) then good growth on acetate, acetate and glutamate, and acetate plus glucose was restored. The CS1- cell produced by gene disrup- tion (CITI::LEU2; see “Materials and Methods” did not con- tain CS protein as determined by immunoblotting (see below).

Respiration of Citrate Synthase Deletion Mutants-Under the same conditions that were used in the growth experiments but with a soft agar overlay containing tetrazolium to measure respiration, only the organisms with CSl, (parental strain, CS2-, and CSl-+YCSl/pFL-l) gave positive results (color change) on acetate + glucose. Since this method is not quan- titative, the rate of respiration was determined with a Clark electrode (Table 111). The respiration of CS1- cells did not differ from either parental or CS2- cells when grown on galactose. No increase in respiration was found in CS1- cells following overnight incubation in acetate medium, whereas parental and CS2- cells responded to acetate incubation with a 4- and 6-fold increase in respiration, respectively.

Enzyme Content of Citrate Synthase Mutants-The strains showed the expected changes in citrate synthase content (Table IV). Cells lacking CS1 (CSl-+pK10) showed only the expected CS2 level of activity in galactose grown cells (20 milliunits/mg). As expected this activity was repressed (<5 milliunits/mg) when the cells were grown on glucose. Intro- duction of cloned CS2 (CSl-+YCSZ/pKlO) and CS1 (CSl-+YCSl/pFL-l) genes into the cells produced increases in citrate synthase activity in galactose grown cells, which

TABLE 111 Respiration of galactose-grown PSY142parenta1,

Csl-, and Cs2- c e h Respiration was measured with an 0, electrode on cells grown on

complete medium with galactose and cells grown on galactose and incubated overnight in complete medium with acetate. The viability of cells was measured after the acetate incubation and was greater than 90%. The medium for the determination of respiration was the same as the growth medium. Values represent duplicate determina- tions on two different cultures f S.D.

Strain Galactose Acetate“ nmol 02/mg proteinlmin

16 k 3 63 f 8 13 f 3 12 f 3 13 f 2 80 f 10

Parental cs1- cs2-

Incubated overnight on acetate.

TABLE JV Expression of yeast CSl and yeast CS2 in

P S Y I 4 2 and its transformants The determination of citrate synthase activity is described under

“Materials and Methods.” Cells were grown in liquid minimal medium containing glucose or galactose and appropriate nutritional factors. Values represent the average of three determinations of two cultures f S.D.

Strain CS activity

Glucose Galactose milliunits/mg protein

Parental CS1-/pK10

35 f 6 <5

90 f I

CSl-/pKCS2 20 f 5

40 f 5 CSl-/pFCSl

90 f 8 80 f I 160 f 8

again was repressed when the cells were grown in glucose. We showed earlier (6) that CS1- cells have altered levels of

a number of enzymes and that CS2- cells have the same enzyme content as the parental cells. We now have extended these studies to include the transformed strains (Table V). For the mitochondrial enzymes of the Krebs tricarboxylic acid cycle, the changes of enzyme activity in the various transform- ants were consistent with our earlier results. In the CS1- derivatives (CSl”kpK10, CSl-+YCSB/pKlO) only the activ- ities of pyruvate dehydrogenase and a-ketoglutarate dehydro- genase complexes were significantly reduced compared with the CS2- derivatives (CS2- and CSl-/pFCSl) that have ac- tivities similar to the parental strain.

We examined also the levels of some enzymes involved in acetate metabolism. Cells grown in galactose were incubated overnight in acetate and various enzymes in the extracts assayed (Table VI). As seen previously, the enzyme content in CS2- was almost the same as the parental strain for four enzymes of the glyoxylate cycle and for two enzymes of gluconeogenesis. In addition, these enzymes were induced by growth on acetate. Deletion of CS1 (CSl-) caused a reduction in the activity of isocitrate lyase, malate synthase, fructose- 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and acetyl-coenzyme A synthetase when the cells were incubated in acetate. Reductions in all these enzymes (except phos- phoenolpyruvate carboxykinase which is undetectable) are seen also in the galactose grown CS1- cells.

Oxidation of Some Tricarboxylic Acid Cycle Intermediates by Mitochondria of CSI- Cells-The oxidation of a-ketoglu- tarate, citrate, and isocitrate was measured in coupled mito- chondria (that is O2 uptake with ADP (state 4) is three to four times that of without ADP (state 3) isolated from paren- tal cells and CS1- cells. These oxidations were compared with NADH oxidation rates. Yeast mitochondria can oxidize ex- ternal NADH with a dehydrogenase located toward the outer surface of the inner membrane (30). Since NADH oxidation rates were unchanged in CSI- mutants, small variations in mitochondrial preparations could be corrected. The results indicated a reduction in the capacity to oxidize these sub- strates by the CS1- mitochondria (Table VII). Reduction in the oxidation of a-ketoglutarate was probably due to the reduced or-ketoglutarate dehydrogenase complex activity (see above). Isocitrate oxidation was reduced to 70% of parental type mitochondria. Citrate oxidation showed the largest de- crease in CS1- mitochondria; it was 37% of the parental type. This decrease could be explained only partially by the decrease in isocitrate dehydrogenase activity. Comparison of citrate oxidation to isocitrate oxidation in the same mitochondria showed that decrease was still 54%. This cannot be explained by the decreased isocitrate dehydrogenase or by the slight decrease in aconitase activity. We observed that oxidation of citrate and isocitrate by sonicated parental mitochondria showed oxidation rates only slightly lower than those of CS1- cells (data not shown).

f3C]Acetate Metabolism-The parental cell line and CS1- and CS2- were grown in complete medium with galactose to mid-growth phase and transferred to minimal medium con- taining 100 mM [2-13C]acetate, pH 5.5, and were incubated for 40 min. There was little difference in the proton-decoupled 13C NMR spectrum of the perchloric acid extracts of the parental (Fig. l a ) and CS2- mutants (b). However, the CS1- mutant showed decreased incorporation of 13C into glutamate (less than 50% of the incorporation in parental or Cs2-, data not shown), a substantial increase in 13C incorporation into aspartate, and a shift in the relative intensities of the gluta- mate multiplets ( c ) . If flux through malate synthase from

Metabolism of Yeast Citrate Synthase Mutants TABLE V

Activities of enzymes in yeast citrate synthase mutants PSY142 cells were grown in liquid minimal medium containing galactose supplemented with lysine and leucine.

Mitochondria were prepared as described under “Materials and Methods.” The superantant solution obtained from the mitochondrial preparation was used to measure the cytosolic enzyme activities. Values represent the average of three determinations from two cultures f S.D. CS, citrate synthase; ICDH(NAD’), isocitrate dehydrogenase (NAD+); a-KGDC, a-ketoglutarate dehydrogenase complex; PDC, pyruvate dehydrogenase complex; SDH, succi- nate dehydrogenase; FUM, fumarase; MDH, malate dehydrogenase; NADH-cytc oxidase, NADH cytochrome c oxidase; cytc oxidase, cytochrome c oxidase; CAT, carnitine acetyltransferase; ADH, alcohol dehydrogenase; GDH, glutamate dehvdroaenase.

11207

Mitochondrial

c s2 - enzymes

CSl-/pFCSl CSl-/pKCS CSl-/pK10

Cytosolic enzymes

c s2 - CSl-/pFCSl CSl-/pKCS2 CSl-/pKlO

.NAD+) ICDH NADH- MDH FUM SDH PDC a-KGDC

.” . oxida- .

rnilliunits/rng protein I

9 0 0 f 1 0 850 f 10

4 0 0 f 5 0

400 f 40 <5 3 9 0 f 40 <5 450 f 45

<5

8 5 f 3

-

- 1121

- 1 5 f l -

130 f 130 f 120 f 110 f

85 f 12 80 f 10 40 f 45 f 7 b

120 f 8 110 k 7 80 f Ib 75 f 7b

__ cytc. se

20 f 2 20 f 1

1600 f 150

1600 f 120 20 k 1 1500 f 140 20 k 1 1700 f 120

- - - - - - - -

6000 f 100 6500 f 100 6000 f 100 5000 f 100

- - - -

I

140 f 12 136 f 10 120 f 10 128 f 13

A dash indicates the enzyme was not measured. Indicates significant difference from CS2- (and parental) levels at p < 0.05.

TABLE VI Enzyme actiuities of PSY142 and its CSI- and CS2- derivatives Viability of CS1- cells after incubation in acetate medium was

90%. Preparation of cell-free extracts for enzyme assays was done as described under “Materials and Methods.” Values represent the av- erage of duplicate determinations of three cultures f S.D. ICL, isocitrate lyase; MS, malate synthase; FDPase, fructose 1,6-bisphos- phatase; PEPCK, phosphoenolpyruvate carboxykinase; Ac-CoA syn., acetyl coenzyme A synthase; CS, citrate synthase.

Enzyme Strain Activity

Galactose Acetate”

ICL Parental cs1- cs2-

MS Parental cs1- cs2-

FDPase Parental cs1- cs2-

PEPCK Parental cs1- cs2-

Ac-CoA syn. Parental cs1- cs2-

cs Parental cs1- cs2-

rnilliunits/mg 14.5 f 4.5 256 k 56 13.8 f 2.0 104 f 9 16.8 f 6.8 254 f 45

15.0 * 4.1 318 f 68 8.5 * 4.6 235 f 38

22.0 f 8.0 308 5 58

18.8 f 5.6 57 * 13 7.4 f 3.3 31 & 5

14.4 f 3.5 55 +- 19

0 0

96 f 14

0 25 f 6.5

108 f 15

55 f 5 116 f 4 44 f 3 48 f 7 57 f 1.5 141 2 8

216 & 20 919 f 32 9 + - 2 85 f 14

378 * 28 808 f 26 “Cells were grown in complete medium with galactose and then

incubated overnight in complete medium with acetate.

glyoxalate is low relative to citrate synthase flux, then analysis of these multiplets is feasible and indicates that the fractional enrichment of acetyl-coenzyme A feeding the citric acid cycle is about 80% in all three cases (31). The difference in the relative intensities of the glutamate multiplets in CS1- com- pared with the parental strain is due to a 4-fold (16-61%)

idase ADH

- -

- -

4000 f 200 4500 f 250 4000 f 200 3900 f 200

GDH

0 0 0 0

26 f 1 23 f 2

16.0 f 1 16.0 f 1

increase in the flux of unlabeled carbon into the citric acid cycle relative to citrate synthase. Decreased incorporation into glucose was also observed whereas little or no changes of incorporation into ketone bodies was seen (data not shown). The reduction in incorporation of 13C from acetate into glu- cose by CS1- was probably due to the reduction in the gluco- neogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose 1,6-bisphosphatase (see above). The increased incor- poration into aspartate might be the result of greatly reduced glucose biosynthesis with only slight reduction in the activity of the glyoxylate cycle in these galactose grown cells.

The Effect of Inactive Citrate Synthase on the CSl- Phe- notype-The CS1- phenotype is characterized by the inability to grow on acetate. CS1- cells transformed with the plasmid YEp352/CSl produced by citrate synthase (Table VIII) which was located in the mitochondria (Table IX) and regained the ability to grow on acetate. When CS1- cells were transformed with either YEp352/CSl/D414G or YEp352/CSl/H313G in which the essential Asp414 (D414) or His313 (H313) were modified to Gly, the CS1 protein was expressed and trans- ported to the mitochondrion as shown by Western blot analy- sis of mitochondrial extracts (Fig. 2). Both Asp414 and His313 were shown to be essential for the catalytic reaction of yeast CS1 since substitution of either of these residues with Gly produced inactive enzyme (Tables VI11 and IX). The citrate synthase activity, however, in whole cell extracts of these cells, was the same as in cells transformed with YEp352 alone (Table VIII) and represented the low CS2 activity present in CS1- cells. This finding was confirmed by showing that the isolated mitochondria of these cells had no detectable citrate sythase activity (Table IX). Note that the low quantity of CS2 protein in CS1- cells as well as lower reactivity with anti- citrate synthase antibody yielded no protein band correspond- ing to CS2 in the immunoblots (Fig. 2 ) . Therefore, both CS1 substitution mutations, Asp414 to Gly (D414G) and His313 to Gly (H313G), expressed enzymatically inactive but struc- turally intact citrate synthase molecules.

To test the hypothesis that the structural integrity of me- tabolon components is important in sequential reactions, we analyzed the level of other Krebs tricarboxylic acid cycle

Metabolism of Yeast Citrate Synthase Mutants

TABLE VI1 0, consumption of coupled PSYI42 and CS1- mitochondria

Oxygen consumption of mitochondria isolated from parental and CS1- strains was measured as described under “Materials and Methods.” RCR (respiration control ratio) is the ratio of respiration measured in state 3 and state 4 on NADH as substrate. The concentrations of a-ketoglutarate (a-KG), citrate (Cit), and isocitrate (Isocit) were 5 mM and NADH was 0.5 mM. Oxidation ratios are the ratio of 0 2 consumption obtained on the substrates indicated. Values are the average of trblicate determinations of two different DreDarations f S.D.

RCR QO, (NADH) Oxidation ratio

a-KG/NADH Cit/NADH Isocit/NADH Isocit/Cit nmol 02/mg min

Parental 4.2 f 0.5 250 f 20 0.2 f 0.01 0.4 f 0.02 0.2 f 0.002 0.5 cs1- 4.0 f 0.4 300 f 25 0.09 f 0.01 0.15 f 0.04 0.14 f 0.06 0.93

a.

b. I

C. A3

I I GN4

I , , , I , , ! 1 1 1 1

3 5 3 0 25 PPM FIG. 1. Proton-decoupled ‘’C NMR spectrum (expanded) of

perchloric acid extracts. Parental ( a ) , CS2- ( b ) , and CS1- ( c ) were grown in [2-13C]acetate for 40 min as described under “Materials and Methods.” Resonance assignments: A3, aspartate C3; G4, glutamate C4; GN4, glutamine C4; and G3, glutamate C3. Spectrum ( c ) is run at a higher sensitivity than spectra a and b.

TABLE VI11 Citrate synthase activity of PSY142 CSI- transformants

grown in glucose or acetate PSY142 CS1- cells were transformed with YEp352 plasmid, with

YEp352 carrying CS1 (YEp352/CS1), or with YEp352 carrying either D414G or H313G mutant CS1 genes (CSl/D414G, CSl/H313G), and cell extracts assayed as described under “Materials and Methods.” Values are means of triplicate determinations of two cultures f S.D. when grown in indicated supplement. ND, not determined since these cells do not mow in acetate.

Plasmid inserted in CS1- Citrate synthase

Glucose Acetate unitslmg

YEp352 0.01 f 0.002 ND YEp352/CSl 1.1 f 0.2 2.2 f 0.2 YEp352/CSl/D414G 0.008 f 0.002 ND YEp352/CSl/H313G 0.012 f 0.002 0.05 f 0.01

TABLE IX Enzyme activities of mitochondria isolated

from PSY142 CSI- transformants PSY142 CS1- cells were transformed with the indicated plasmids.

Mitochondria were prepared, and the enzyme activities were meas- ured as described previously. Values are the means of three determi- nations f S.D. a-KGDC, a-ketoglutarate dehydrogenase complex; PDC, pyruvate dehydrogenase complex; MDH, malate dehydrogen- ase; CS, citrate synthase.

Plasmid inserted in CS1-

Activity

a-KGDC PDC MDH cs unitsjmg

YEp352 0.03f0.01 0.03f0.01 5.0f0.2 C0.005 YEp352/CSl 0.14f0.01 0.13f0.01 5.8f0.2 2.4f0.3 YEp352/CSl/ 0.07f0.005 0.03f0.01 4.9f0.2 C0.005

YEp352/CSl/ 0.12f0.005 0.04f0.01 5.1f0.1 C0.005 D414G

H313G

enzymes (Table IX) and the rate of citrate oxidation (Table X) in mitochondria isolated from these strains. The a-keto- glutarate dehydrogenase complex activity in CSl/H313G- containing cells was nearly the parental level, while the CS1/ D414G-containing cells exhibited only a small increase over background level. The pyruvate dehydrogenase complex level was unchanged in cells containing either mutation.

CS1- cells carrying CSl/H313G grew on plates with acetate as the sole carbon source (Fig. 3). The doubling time of CS1- + YEp352/CS1 was 5 & 0.2 h and that of CS1- + YEp352/ CSl/H313G was 13.6 f 2 h in complete medium containing acetate. The cell transformed with YEp352/CSl/D414G did not grow on acetate. Both substitution derivatives increased the rate of citrate oxidation with respect to isocitrate (Table X).

DISCUSSION

The glyoxylate cycle converts two acetyl coenzyme As to succinate and NADH, and its occurrence in cells that depend on C2 compounds for growth supports the hypothesis that this overall reaction is the chief function of the pathway. Although it is true that several intermediates of the Krebs tricarboxylic acid cycle can be utilized either for protein or carbohydrate synthesis, such carbon utilization must ulti- mately come at the expense of other carbon precursors such as the succinate produced by the glyoxylate cycle. Walsh et al. (32) found in E. coli that contained different levels of citrate synthase, the level of ICDH activity was regulated both by the amount of citrate synthase as well as by the phosphorylation state of ICDH. These changes were accom- panied by changes in metabolite levels and flux.

A phenotypic difference was observed between CS2- and CS1- yeast. In CS2- cells, no changes in the growth pattern, enzyme levels, metabolic behavior, or metabolite levels were

Metabolism of Yeast Citrate Synthase Mutants 11209

TABLE X Respiration of mitochondria isolated from PSY142 CS1- transformants

PSY142 CS1- cells were transformed with the indicated plasmids. The concentrations of isocitrate (isocit), citrate (cit) were 5 mM and that of NADH was 0.5 mM. RCR (respiratory control ratio) is the ratio of respiration measured in state 3 and in state 4 with NADH as substrate. Values are the average of triplicate determinations of two different DreDarations f S.D.

Plasmid inserted in cs1-

Oxidation ratios RCR 02 (NADH) Iswit/

NADH Cit/

NADH Iswit/

Cit

nmol Oz/min/mg protein

YEp352 2.3 f 0.05 275 f 15 0.15 & 0.01 0.16 f 0.02 0.93 YEp352/CSl 2.1 f 0.05 280 & 10 0.16 f 0.01 0.32 & 0.02 0.50 YEp352/CSl/D414G 2.3 f 0.07 290 f IO 0.14 k 0.01 0.27 f 0.03 0.52 YEp352/CSl/H313G 2.3 f 0.05 290 f 10 0.17 f 0.03 0.33 f 0.07 0.51

1 2 3 4

FIG. 3. PSY142 CS1- derivatives were grown on complete medium with acetate. The derivatives containedpanel 1, YEp352/ CSI; panel 2, YEp352/CSI/H313G; panel 3, YEp352/CSl/D414G; panel 4, YEp352.

FIG. 2. PSY142 CS1- derivatives were grown on complete medium with galactose. Mitochondria from the transformants were isolated and subjected to Western blot analysis. The transform- ants contained lane 1 , YEp352/CSl; lane 2, YEp352; lnne 3, YEp352/ CSl/H313G; lane 4, YEp352/CSl/D414G.

detected. The CS2- strain grew on acetate, therefore, no essential metabolic role could be assigned to CS2. CS1- cells, however, did not grow on acetate. This indicated that the citrate produced in the cytosol by CS2 could not be utilized efficiently by the remaining enzymes of the Krebs tricarbox- ylic acid cycle in the CS1- mitochondria. Growth on acetate was restored by reintroducing the CS1 gene in CS1- cells showing that this difference in phenotype was the result of the absence of CS1 enzyme and not a secondary effect.

Additional differences between CS1- and CS2- cells were observed in enzyme and metabolite patterns. Previously (6) it was shown that CS1- cells contained lower a-ketoglutarate and pyruvate dehydrogenase complex activities, and these experiments demonstrate that these activities can be restored by the CS1 enzyme. Deletion of CS1 reduced the induction of isocitrate lyase, fructose-1,6-bisphosphatase, phosphoenolpy- ruvate carboxykinase, and acetyl-coenzyme A synthetase by acetate.

Isolated mitochondria from CS1- cells also revealed a de- creased capacity to oxidize citrate. The decreased [ 13C]acetate incorporation into glutamate suggested a reduced activity of the tricarboxylic acid cycle by CS1- cells. The reduced incor-

poration of into glucose may be explained by the decreased activity of the glyoxylate shunt and gluconeogenic enzymes. The increased incorporation into aspartate in CS1- cells indicated a substantial shift in distribution of carbon in the citric acid cycle and exchangeable pools. The shift in gluta- mate multiplet intensities may be attributed to a 4-fold de- crease in citrate synthase flux. If flux through uninvolved spans of the citric acid cycle is unaffected by CS1- mutation (e.g. for gluconeogenesis), then the observed 5-fold decrease in respiration of CS1- (Table 111) is consistent with these NMR observations. Furthermore, none of the above changes could be attributed to the absence of mitochondrial citrate since CS1- cells contain citrate. Both CS2 activity and a citrate uptake system in the mitochondria of CS1- cells are present.

In order to determine that the reduced mitochondrial citrate oxidation by CS1- cells was due to the deletion of CS1, we constructed mutant CS1 genes. Aspartate 414 (D414) and histidine 313 (H313) are the putative active site residues of yeast CS1 analogous to the active site residues of pig heart citrate synthase aspartate 375 (D375) and histidine 274 (H274) that have been identified by x-ray crystallography (34). These residues are conserved in citrate synthase from several different sources (33) and in these studies were shown to be essential for yeast CS1 activity. When the plasmid containing the CS1 gene, with its signal sequence and muta- genized amino acid sequence, was inserted into CS1- cells,

11210 Metabolism of Yeast Citrate Synthase Mutants

the mutant enzymes were expressed and transported into the mitochondria. The total citrate synthase activity observed in these transformed cells was not more than CS1- cells carrying no CS1 protein, and the isolated mitochondria showed no detectable citrate synthase activity. However, the mitochon- dria were shown to contain CS1 protein by immunoblot techniques. The mitochondria of both mutants had an in- creased rate of citrate oxidation with respect to isocitrate, approaching the value of the parental cells and CS1- cells transformed with YEp352 containing CS1. Both CSl/H313G and CSl/D414G cells showed an increase in a-ketoglutarate dehydrogenase complex activity compared with CS1- cells. No increase in pyruvate dehydrogenase complex activity was observed in either the CSl/H313G- or CSl/D414G-contain- ing cells. In addition, the CS1- cells transformed with CS1/ H313G enzyme grew on acetate, although at a rate slower than the control CS1- cells containing active CS1. CSl/ D414G did not restore growth on acetate to CS1- cells. These observations suggest that the CS1 enzyme may have functions other than its catalytic activity and that the two mutant CS1 proteins may have different final protein structures. I t is possible that the presence of an intact (though inactive) citrate synthase molecule of normal conformation permits the formation of a Krebs tricarboxylic acid cycle complex (35) in which the a-ketoglutarate dehydrogenase complex activity is maintained. Thus, the citrate produced by the cytosolic citrate synthase (CS2) may be more efficiently utilized in the mito- chondrion providing sufficient energy for the cells to grow on acetate.

The importance of protein-protein interactions in metabo- lism and protein expression has been shown for influenza virus hemagglutinin-induced synthesis of glucose-regulated proteins (36) and in mutants in different subunits of the multisubunit complex, coenzyme QH2-cytochrome c reductase (37). These results further support the conclusion that mul- tifunctional complex formation is dependent upon its constit- uent members and the assembly process. We are continuing our studies of these metabolic interrelations and assessing the role of putative metabolic complexes in situ.

Acknowledgments-We are grateful to Dr. L. Guarente for the supply of the CS- mutant cell lines and the YEp352 yeast vector. We thank Dr. G. Schatz for the CS1 gene and the pKlO vector. We appreciate the excellent technical assistance of Ginny Poffenberger and the secretarial skills of Penny Kerby. We also thank Dr. Sarah McIntire for her critical reading of the manuscript.

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