An acyl-thioesterase from yeast mitochondria

ARCHIVESOF BIOCHEMISTRY Vol. 225, No. 2, September,

AND BIOPHYSICS

pp. ‘704-712, 1983

An Acyl-Thioesterase

from Yeast Mitochondria

R. STACK, S. SCHARF, J. B. OHLROGGE,

AND

R. S. CRIDDLE’

Department of Biochemistry and Biophysics, University of Cal$n-nia, Davis, California Received January

95616

31, 1983, and in revised form May 11, 1983

A previously unstudied acyl-coenzyme A thioesterase activity has been demonstrated in submitochondrial particles from Sacch,aromyces cerevisiae. The preferred substrate for the enzyme activity is oleoyl-coenzyme A. Tests with inhibitors of the thioesterase showed that, in addition to common thiol inhibitors, the oxidative phosphorylation inhibitors oligomycin and venturicidin also blocked thioesterase activity. Purification of the enzyme catalyzing this activity revealed that thioesterase copurified with mitochondrial ATPase. When thioesterase was isolated from oxidative phosphorylation mutants selected for resistance to these two inhibitors, thioesterase activity was also resistant. The results suggest that thioester hydrolysis may be catalyzed by components associated with the isolated ATPase complex. Further attempts to link this activity to in viva function of ATPase were not successful.

A number of reports have documented protein thiol involvement in mitochondrial functions (1, 2). Some thiol-dependent mitochondrial enzyme activities have been well characterized and the thiol involvement in the active sites defined. The roles of other thiol-dependent activities have only been inferred mainly from inhibitor studies. Several energy-linked processes of oxidative phosphorylation, for example, are blocked by standard thiol inhibitors but the mechanism and sites of action are unclear (3, 4). These findings led us to investigate some thiol-related catalytic properties of mitochondrial fractions. In particular, hydrolysis of acyl-coenzyme A by mitochondrial preparations was examined. An active acylcoenzyme A thioesterase was solubilized and purified from inner mitochondrial membranes of yeast. Maximum yield of enzyme activity was obtained using procedures commonly employed for extraction and purification of the membrane-bound yeast mitochondrial ATPase complex. Earlier findings of acyl-CoA hydrolase ac’ To whom correspondence

should be addressed.

0003-9861183$3.00 Copyright All rights

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

704

tivities located in the matrix fractions of mammalian mitochondria have been reported (5). Efforts to separate thioesterase from ATPase activity were not successful. Evidence to date, based on inhibition studies and analysis of yeast mitochondrial mutants, suggests that there may be a direct association among at least some of the components responsible for these two activities. However, no evidence for involvement of thioesterase in energy-linked steps of oxidative phosphorylation could be demonstrated. MATERIALS

AND

METHODS

Thioesterase and ATPase were prepared from the yeast Socchorom~ces cererris~ strains D243-4A (a, ade-, lys-), OR4 (an oligomycin-resistant mitochondrial mutant prepared from D243-4A (5)), L4490 (a, ade-, trp-, lys-, oli-r, vent-r), and L748 ((Y, ma-, his-, oli-r, vent-r), a temperature-sensitive venturicidin resistant mutant with the same parent as L411, all supplied by Dr. A. W. Linnane (6), or NE15 (oli-r, vent-r) supplied by Dr. D. E. Griffiths. Nicotinamide adenine dinucleotide (reduced), adenosine triphosphate, adenosine diphosphate, phosphoenolpyruvate, paminobenzamidine HCl, phenyl-

MITOCHONDRIAL

methylsulfonyl fluoride, N-ethylmaleimide (NEM), dithiobis-nitrobenzoic acid (DTNB), and pyruvate kinase were obtained from Sigma Chemical Company. Oligomycin and lactate dehydrogenase were obtained from Calbiochem. Sepharose 6B was obtained from Pharmacia Corporation, Biobeads SM-2 was from BioRad, and Triton X-100 was from Packard Instrument Company. Yeast extract and Bactopeptone were from Difco. [‘“C]Oleoyl-coenzyme A and [3H]oleic acid were from New England Nuclear. Palmitoyl-coenzyme A and stearoyl-coenzyme A were gifts from Dr. P. K. Stumpf. Venturicidin was a gift from Dr. D. E. Griffiths. Yeast were grown in batch cultures at 30°C in a New Brunswick Scientific 20-liter fermentor with vigorous aeration. Media contained 1% yeast extract, 1% bactopeptone, and 2% ethanol. Cultures were harvested at late log phase. Cells were washed twice in breaking buffer and broken using a Braun glass bead homogenizer as described previously (7). Yeast mitochondria and submitochondrial particles were isolated and thioesterase was extracted with peroxide-free Triton X-100. Purification of thioesterase followed a modification of the method used by Tzagaloff (8,9) for purification of oligomyein-sensitive ATPase. Thioesterase was also purified from Triton X-lOO/deoxyeholate extracts using the ATPase purification method of Ryrie (10). Enzymes were incorporated into liposome vesicles as described by Eytan et al. (ll), and Triton X-100 was removed from the protein-liposome vesicles by passage through a Biobeads SM-2 column (12). Protein concentrations were determined by either the Folin (13) or Coomassie blue dye-binding method (14). Acyl-thioesterase activity was assayed by the method of Shine et al (15). ATPase activity was assayed either by the coupled pyruvate kinase-lactic dehydrogenase reaction as described by Munroy and Pullman (16) or by the measurement of inorganic phosphate according to Lowry and Lopez (17) when in the presence of thiol inhibitors. N-ethylmalemide inhibition utilized the method of Riordan and Vallee (18). Lipophilic inhibitors were added as either dimethyl formamide or methanolic solutions. Peroxide-free Triton X-100 was prepared by Naz&Or reduction following an initial titration of a small aliquot of a 10% solution to an endpoint determined with KI. This solution was then stored at 4°C under either nitrogen or argon. SDS-gel electrophoresis was carried out on 7.5% gels using the method of Laemmli and Favre (19). ’ Abbreviations used: NEM, N-ethylmaleimide; DTNB, dithiobis-nitrobenzoic acid; SDS, sodium dodecyl sulfate; SMP, submitochondrial particles; DTT, dithiothreitol; CCCP, carbonylcyanide m-chlorophenylhydrazone; DCCD, dicyclohexylcarbodiimide.

705

THIOESTERASE RESULTS

An acyl-thioesterase activity, catalyzing hydrolysis of long-chain acyl-coenzyme A derivatives, can readily be detected in preparations of isolated (broken) yeast mitochondria. Thioesterase activity in the mitochondrial preparation is predominantly membrane bound; greater than 75% of the activity remains associated with submitochondrial particles (SMP) after mild sonication (1 min in a Bransonicbath sonicator) and centrifugation. Parallel measurements, for comparison, indicated that near 85% of the mitochondrial ATPase activity also remained bound to SMP. Membrane-bound thioesterase could subsequently be solubilized with detergents. A single extraction of SMP which 0.1% Triton X-100 typically solubilized 2040% of the bound thioesterase activity and about 45% of the ATPase activity. Approximately 30% of the bound thioesterase activity remained unextractable even after repeated Triton X-100 treatments. Figure 1 illustrates the elution profiles of protein, thioesterase, and ATPase activities in the Triton X-lOO-solubilized extracts of SMP following chromatography on Sepharose 6B (9). Three major protein peaks are commonly observed, with peak II containing both the ATPase and thioesterase activities. Occasionally some thioesterase activity eluted with peak III. This was never more than 5-10% of that found in peak II and was not characterized further. Alternative, but not greatly dissimilar, published methods for purification of the oligomycin-sensitive ATPase all yielded enzyme catalyzing thioester hydrolysis. In all cases similar thioesterase/ATPase activity ratios were observed. These methods included the glycerol gradient centrifugation method of Tzagoloff (20), the methods of Enns and Criddle (9), and the method of Ryrie (10). Incorporation of purified protein preparations with both ATPase and thioesterase activity into lipid vesicles (11) followed by chromatography through Biobeads SM2 to remove Triton X-100 (12), resulted in both activities becoming vesicle bound. A

706

STACK

ET AL.

? z

6.0

3.0

4.0

2.0

5

B

0 0 z G 1.0

2.0

G 5

L 10

30

20

FRACTION

40

50

NUMBER

FIG. 1. Concentrated Triton X-100 extract (2.0 ml) prepared from yeast submitocbondrial particles as described (9) was cbromatographed on a 2 X 45-cm column of Sepharose 6-B. Four-milliliter fractions were collected and aliquots were assayed for ATPase activity(X) (16), thioesterase activity (0) (15), and protein (0) (14).

two- to threefold increase in specific activities of both ATP and oleoyl-coenzyme A hydrolyses was noted. Typical results are summarized in Table I. Electrophoretic analysis of purified enzyme preparations having both ATPase and thioesterase activities showed only one major protein band on native gels. Figure 2 shows the distribution of subunits obtained following SDS-gel electrophoresis of such preparations. In all studies the TABLE

commonly observed ATPase bands were present, and only minor, variable amounts of other protein components. Figure 3 presents kinetic characteristics of thioester hydrolysis catalyzed by purified thioesterase. Figure 3A illustrates the change in reaction velocity with change in oleoyl-coenzyme A concentration. Halfmaximal velocity is observed in the substrate concentration range of 2-3 PM. This is not likely to represent a “true” Michaelis I

PURIFICATION OF ATPase AND THIOESTERASE ACTIVITIES FROM YEAST MITOCHONDRIA Ratio ATPase

Thioesterase Activitya Triton extract Glycerol gradient Sepharose 6B column Liposome-bound enzyme

Activity”

% Recovery

% Recovery

ATPase/ thioesterase

0.38 0.78 1.1

100 49 27

11.5 26.7 38.0

100 41 32

33 35 25

3.2

-

86.5

-

27

DValues given as pmol substrate

hydrolyzed/min/mg

protein

at 37°C.

MITOCHONDRIAL

FIG. 2. Electrophoresis of a purified protein preparation (9) containing both ATPase and thioesterase activities. Electrophoresis was performed in 7.5% polyacrylamide gels by the method of Laemmli and Favre (19).

kinetic constant owing to the possibility of micelle and mixed micelle formation by the substrate. Figure 3B demonstrates reaction rate dependence on enzyme concentration. A linear relationship between added enzyme and rate is observed at low levels of enzyme. The nonlinearity seen at higher concentrations may result from detergentlike interactions of substrate with enzyme and/or effects of Triton on substrate solubilities. Figure 3C shows oleoyl-coenzyme A hydrolysis with respect to time. Linearity is observed for the first 4 to 5 min after addition of enzyme with the conditions employed. Figure 3D displays activity dependence on pH over the range 6-9. Nearmaximal activity is observed over a broad pH range centered around pH 7.5. Endproduct inhibition was not observed upon preincubation with either oleic acid or coenzyme A. The temperature dependence of purified thioesterase before and after reconstitution into liposomes was examined. An Arrhenius plot of the data is shown in Fig. 4. The reconstituted enzyme displayed a break in the Arrhenius plot at 16-17’C, a discontinuity not observed for the deter-

THIOESTERASE

707

gent-solubilized enzyme. ATPase activity for enzyme bound to liposomes also showed a similar thermal transition not observed for the detergent-solubilized ATPase. A limited test of substrate specificity showed that thioesterase functions most effectively with oleoyl-coenzyme A. This activity is at least eightfold faster than hydrolysis of either stearoyl or palmitoylcoenzyme A (Table II). Thin-layer chromatography confirmed the identity of oleic acid as a reaction product. Thioesterase activity was examined in the presence of inhibitors of oxidative phosphorylation (Table III). Dinitrophenol, 1799 (bis-hexafluoroacetonyl acetone), and azide had no effect on thioesterase activity. Carbonylcyanide m-chlorophenylhydrazone (CCCP) caused a slight inhibition. Valinomycin stimulated activity slightly. Dicyclohexylcarbodiimide (DCCD), which blocks ATPase activity by reaction with the proteolipid subunit 9 (21), had no effect on thioesterase activity. Bicarbonate, a reagent known to stimulate ATP hydrolysis (22), also had no effect on thioesterase. The mitochondrial thioesterase appears to be a thiol-dependent enzyme. Thus, enhanced recovery of enzyme activity was afforded by the inclusion of DTT in purification buffers and by the use of peroxidefree Triton X-100 during extraction procedures. Reagents primarily affecting thiol groups were efficient inhibitors of thioesterase. Iodoacetamide, NEM, DTNB, and Pb(NO& all inhibited thioesterase activity. Mercuric ion is a particularly potent inhibitor of thioesterase with 50% inhibition noted with 100 nM Hgz+. This level of Hg2+ has little effect on the ATPase activity in the same preparation of isolated enzyme. Titration curves showing inhibition of ATPase and thioesterase activities with Hgz+ are shown in Fig. 5A. Figure 5B shows similar inhibition curves demonstrating the effect of NEM on these two activities. Again, inhibitor concentrations sufficient to block greater than 90% of thioesterase activity have little effect on ATPase activity. Other workers have also reported the relative insensitivity of ATPase to thiol inhibitors. However, an extreme sensitivity of the ATPase catalyzed ATP-Pi exchange

708

STACK

ET AL.

B.

ENZYME

CONC.

(MIIII)

ii s0

ii 3 -0 w g 4.0

y 0.45

P r B 2 0.40

t - 3.0 z a 0 g

6 a 2.0

p 0.35

8 01

TIME

(minutes)

FIG. 3. Kinetics of acyl-thioesterase reactions catalyzed by ATPase preparations. (A) Velocity of the reaction at various substrate concentrations. A 1.4~rg amount of Sepharose-purified protein was assayed for 5 min. (B) Velocity of the reaction with respect to the amount of added enzyme. Substrate concentration [l’C]oleoyl-coenzyme A, 2 X 10’ dpm/nmol) was maintained at 10 pM. (C) Velocity of the thioesterase reaction with respect to time. Both the amount of enzyme (1.0 pg) and substrate (10 prd [“Cjoleoyl-coenzyme A, 2 X 10’ dpm/nmol) were maintained as constant. (D) The pH dependence of thioesterase using [14C]oleoyl-coenzyme A as substrate. Reaction rates using 0.42 pg of reconstituted enzyme were measured in 50 mM Mes (pH 6.0-6.5), 50 mM Mops (pH 7.0-7.5), or 50 mM Tris (pH 7.5-9.0) containing 200 mM sucrose and allowed to proceed for 1 min.

to the same agents is commonly noted (3, 4). Two specific inhibitors of mitochondrial ATPase activity, oligomycin and venturicidin, are shown in Table IV to also cause significant inhibition of thioesterase activity. Thioesterase prepared from strain D243-4A, a wild-type strain in which

ATPase activity is both oligomycin- and venturicidin-sensitive (O’V’), is both oligomycin- and venturicidin-sensitive as well. The thioesterase from strain OR4,an oligomycin-resistant mitochondrial gene mutant (ORVS) derived from D243-4A, showed little or no change in resistance to either of these inhibitors when compared

MITOCHONDRIAL

g x-

709

THIOESTERASE TABLE

3.1 3.0 2.9 2.8 2.7 2.6 i 2.5 2.4 23 L-

III

“.

EFFECB INHIBITORS

I

32

,

3.4

3.3

3.5

1

36

to the parent strain. In contrast, when strain L411 (OsVs) is compared with the related mutant strain L748 (ORVR),thioesterase sensitivity to oligomycin is low but unchanged, while sensitivity to venturicidin is virtually eliminated in the mutant strain. Two additional mutant strains, L4400 and NE15 (ORVR),were studied. No parent strain was available for direct comparison in these cases, but L4400 thioesterase activity showed substantial sensitivity to oligomycin while neither strain was inhibited by venturicidin at levels found to inhibit D243. Oligomycin and venturicidin are not general inhibitors of thioesterase activities as they have no effect on the activity of a plant thioesterase (23) isolated from avocado (data not shown). DISCUSSION

A novel acyl-coenzyme A thioesterase has been solubilized from mitochondrial

SUBSTRATE

II

SPECIFICITY

Substrate

Level

pmol/min

1610 CoA 16:0 CoA

2 IrM 20 /AM


18:O CoA 180 CoA

2 PM 20 FM

0.13
18:l CoA 181 CoA

2 PM

9.5

20 /AM

13.0

PHOSPHORYLATION

Inhibitor

Concentration

% Inhibition

Dinitrophenol CCCP Sodium azide 1799 DCCD Valinomycin NaHCO,

20 pg/ml 10 rg/mi

0 8 0 0 0 (+17%) 4

37

FIG. 4. Arrhenius plot of thioesterase kinetics. Soluble enzyme purified through Sepharose 6-B (1.3 pg) and reconstituted enzyme (0.8 pg) were assayed at different temperatures. The substrate was 5.0 PM [Woleoyi-coenzyme A. Reconstituted enzyme is shown as (0) and soluble enzyme as (0).

TABLE

OF OXIDATIVE

ON THIOESTERASE ACTIVITY OF ATPase ISOLATED FROM STRAIN D243-4A

3mM

10 10 2 10

@g/ml pg/ml @g/ml rnM

membranes of S. cerevisae. Three isolation procedures yielded preparations with common properties. In each case, thioesterase copurified with mitochondrial ATPase. The copurification data suggest that the two enzymes have similar physical properties or are associated in some fashion, possibly even to the extent of being parts of the same enzyme complex. Copurification does not establish that two enzymes are associated, only that they have similar physical properties. It cannot be concluded that our ATPase preparations are pure or that the methods for preparation of ATPase-thioesterase could not be improved to separate the two activities. Thioesterase could be a common contaminant of each ATPase preparation, possibly identifiable with one of the faint undefined bands observed on SDS-gel electrophoresis of ATPase preparations. Even the observed parallel incorporation of enzymes into liposomes, with proportional increases in specific activity, could be fortuitous. Three lines of evidence beyond the copurification observations require that the possible coexistence of the two enzyme activities within one complex be considered. First, conditions employed for the reconstitution of enzyme into lipid vesicles gives a parallel incorporation of both enzyme activities, and the specific activities of both increase by the same factor. Second, oligomycin and venturicidin, two well-characterized inhibitors of oxidative phosphorylation and ATPase activities, also block thioesterase activity with apparently

710

STACK

I

I

1

I

50 100

250

500

Hg 2 CONCENTRATION

NEM

CONCENTRATION

(nM)

W.4)

FIG. 5. Inhibition

of thioesterase and ATPase activities by thiol reagents. (A) Titration of ATPase activity (3.4 pg of soluble ATPase assayed by measurement of Pi produced (17)) and thioesterase activity (2.0 pg of soluble purified enzyme plus 5.0 PM [l’C]oleoyl-coenzyme A) with HgQ. Results are expressed as the percentage of control activity with no inhibitor. ATPase (0) and thioesterase (0) activities are plotted. (B) Inhibition of both ATPase activity (2 pg, (0)) and thioesterase activity (1 pg protein, (0)) by increasing amounts of N-ethylmaleimide (NEM). Results are expressed as the percentage of activity of controls containing 10 mM DTT and no inhibitor (18).

identical binding constants. Third, mitochondrial gene mutations which confer venturicidin resistance to selected yeast strains by reducing ATPase sensitivity to this antibiotic also caused a reduced sensitivity of thioesterase activity to venturicidin.

ET AL.

Parallel sensitivities to specific oxidative phosphorylation inhibitors for two separate, nonassociated enzymes are, however, more difficult to explain. Some common inhibitor-binding sites must be present. If thioesterase is not associated with the intact ATPase complex, it may be associated with oligomycin- and venturicidin-binding proteins of the ATPase complex. Since oligomycin and venturicidin both inhibit at the level of the F,, portion of ATPase, thioesterase may be bound to the F0 proteins. The association of thioesterase with ATPase or ATPase subunits could also account for the parallel changes in inhibitor sensitivities of enzyme activities upon formation of antibiotic-resistant mutants. As the mutations appear to be at single sites on the mitochondrial genome, simultaneous effects on both enzyme activities can best be accounted for by an association of activities with common subunits. It is well established that the energycoupling reactions of ATPase are highly sensitive to thiol reagents while ATPase activity is not. For example, Boyer et al. (4) observed 50% inhibition of ATP-Pi exchange by 40 j.kM p-mercuribenzoate with no detectable effect on ATP hydrolysis. Sanadi et aL (3) inhibited 85% of the ATPPi exchange catalyzed by mitochondrial membranes using 150 PM p-chloromercuriphenylsulfonate while noting little effect on ATP hydrolysis. Blanchey et al. (24) have concluded that the key thiol group(s) functioning in ATP-Pi exchange is located in the F0 portion of the ATPase complex. Genetic evidence also strongly suggests a role for the M, 20,000 subunit 6 from F,, in this energy-linked process. For example, Roberts et al. (25) have isolated three different mutants of yeast with altered subunit 6 and find no ATP-Pi exchange. Another mutant, mapping at the same locus (oli 2), was able to catalyze ATP-Pi exchange but only at 10% of the wild-type rate, even though an active oligomycin-sensitive ATPase could be demonstrated (26). It is worth noting that venturicidin resistance also maps at the oli 2 locus, which has been shown to code for subunit 6 of the ATPase complex (27).

MITOCHONDRIAL TABLE

711

THIOESTERASE IV

EFFECTS OF OLIGOMYCIN AND VENTURICIDIN ON THIOESTERASE ATIVITIES % Inhibition D243-4A (OSVS)

Strain Oligomycin 20 40 60 200 Venturicidin 10 20 30 50 100

DZ42-4A-OR4 (ORVS)

L411 (OV)

L748 (ORVR)

L4400 (ORVR)

NE15 (ORVR)

&g/ml)” 20 35 53 72

19 19 58

10

18 48

(pg/ml) 5 15 35 60 95

28 32

19 32 38 40

0 +6 0 0

0 +12 0 0

0 0

a Oligomycin inhibition of ATPase enzyme from D243-4A in parallel experiments gave values as follows: 20 pg/ml = 32%, 40 pg/ml = 50%, 60 rg/ml = 85%, and 200 pg/ml = 92% inhibition.

Griddle et al. (28) have reported association of pantothenic acid with yeast mitochondrial ATPase preparations. The pantothenic acid was found to migrate on SDS gels coincident with subunit 6. This thiol could be involved in the observed thioesterase reaction. Since pantothenic acid functions biochemically as an acyl group carrier, this would seem to implicate acyl groups or their derivatives in some as yet undetermined function in this preparation of ATPase. The thioesterase activity studied here may not represent the true activity of this enzyme in viva as thioester hydrolysis can be catalyzed by a number of acyl transfer enzymes. Also the venturicidin-sensitive thioesterase reported here may not be representative of any chemical step functioning directly in ATP synthesis, but could be indicative of events occurring during partial reactions catalyzed by components of the ATPase complex. REFERENCES 1. GRIFFITHS, D. E. (1976) in Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, T., Neupert, W., Sebald, W., and Werner, S., eds.), pp. 175-185, North, Amsterdam.

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GODINOT,

B&hem.

C.,

AND

Biophys.

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GAUTHERON,

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AND

Sot.

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T., PARTIS,

M.,

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