Vol. 133, No. 2

JOUJRNAL OF BACTERIOLOGY, Feb. 1978, p. 584-592 0021-9193/78/0133-0584$02.00/0 Copyright (© 1978 American Society for Microbiology

Printed in U.S.A.

Mitochondrial Adenosine Triphosphatase of Wild-Type and poky Neurospora crassa STANLEY E. MAINZERt AND CAROLYN W. SLAYMAN* Departments of Human Genetics and Physiology, Yale University School of Medicine, New Haven, Connecticut 96510 Received for publication 19 August 1977

We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant known to possess defective mitochondrial ribosomes and reduced amounts of cytochromes aa3 and b. poky contains two distinct forms of mitochondrial ATPase. The first is normal in its Kn. for ATP, specificity for nucleotides and divalent cations, pH optimum, cold stability, and sensitivity to inhibitors (oligomycin, N,N-dicyclohexyl carbodiimide, and adenylyl imidodiphosphate). The fact that membrane-bound, cold-stable, oligomycin-sensitive ATPase activity is present in poky (with an activity of 1.93 ± 0.03 ,umol/min mg of protein compared with 1.33 ± 0.07 ,tmol/min mg of protein in the wild-type strain) and also in chloramphenicol-grown wild-type cells suggests that products of mitochondrial protein synthesis play only a limited role in the attachment of the mitochondrial ATPase to the membrane in Neurospora. poky also contains a second form of mitochondrial ATPase, which has an activity of 1.5 ± 0.2 ,umol/min mg of protein, is oligomycin sensitive but cold labile, and presumably is attached less firmly to the mitochondrial membrane. The two forms, added together, represent a substantial overproduction of mitochondrial ATPase by poky. In an attempt to understand the way in which genes specify mitochondrial proteins, much attention has been focused upon the poky mutant of Neurospora crassa. poky, first described in 1952 by Mitchell and Mitchell (17), is a maternally inherited mutant with a grossly defective cytochrome chain. It contains very little cytochrome aa3 or b, and, as a result, both cyanidesensitive respiration and oxidative phosphorylation are reduced by comparison with wild-type Neurospora (11-14, 43). Instead, poky makes use of a derepressed alternate oxidase that branches from the usual electron transport chain at the level of flavoproteins and-although not coupled directly to any phosphorylation site-permits ATP synthesis by substrate-level phosphorylation and probably by oxidative phosphorylation at site I (33). There is now good evidence that the primary lesion in poky is in the small subunit of mitochondrial ribosomes (9, 10, 26). The resulting deficiency of intact mitochondrial ribosomes leads to a slowing down of mitochondrial protein synthesis and can readily account for the observed abnormalities in cytochrome content, because cytochromes aa3 and b are known to possess mitochondrially synthesized polypeptides (31,

41, 42, 44). The effect of thepoky mutation upon another major mitochondrial enzyme, mitochondrial adenosine triphosphatase (ATPase), has not yet been examined in detail. Experiments with yeast have established that mitochondrial ATPase, like cytochromes aa3 and b, contains multiple polypeptide chains, some of which (the constituents of the Fl portion and stalk) are synthesized in the cytoplasm whereas others (hydrophobic polypeptides tightly associated with the inner membrane) are synthesized in the mitochondria (32, 36, 38, 39). As a consequence, interfering with mitochondrial protein synthesis in yeast (for example, by adding chloramphenicol) results in inhibition of the assembly of the complete oligomycin-sensitive mitochondrial ATPase complex; instead, oligomycin-resistant, cold-labile Fl ATPase activity accumulates in the cytoplasm (35). Similarly, when the synthesis of the mitochondrially made subunits is prevented by mutation, the ATPase becomes oligomycin resistant and can be easily detached from the inner membrane by sonic disruption (8, 22, 28). The extent to which the biogenesis of mitochondrial ATPase in Neurospora (an obligate t Present address: Fundamental Research Unit 6, Coming aerobe) resembles that in yeast (a facultative Glassworks, Sullivan Park, Coniing, NY 14830. anaerobe) is not yet clear. Recently, an inactive 584

VOL. 133, 1978

MITOCHONDRIAL ATPase OF NEUROSPORA CRASSA

ATPase complex has been isolated from detergent-solubilized submitochondrial particles of Neurospora by immunoprecipitation with antibody to purified Neurospora Fl ATPase (6). This complex contains approximately 14 polypeptides, and differential labeling in the presence of chloramphenicol and cyclohexinide indicates that at least 2 of the polypeptides are synthesized mitochondrially. However, one specific ATPase component known to be synthesized mitochondrially in yeast-the membrane proteolipid that binds N,N-dicyclohexyl carbodiimide (DCCD) (32)-appears to be synthesized in the cytoplasm in Neurospora (30). The experiments reported in the present paper were undertaken to characterize the mitochondrial ATPase of Neurospora in its active, membrane-bound forn and to see to what extent the poky mutation leads to defects in the ATPase. A preliminary account of this work has already been published (S. E. Mainzer and C. W. Slayman, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, K15, p. 139). MATERIALS AND METHODS Growth of Neurospora. Wild-type strain RL21a andpoky strain NSX fwere used in these experiments; f is a nuclear-gene suppressor of the poky mutation that increases the growth rate without restoring the wild-type respiratory system (18). Conidia were inoculated at a density of 106 per ml into Vogel minimal medium (40) containing 2% sucrose as the carbon source and were grown with vigorous aeration at 25°C for 15 h (wild type) or 21 h (poky). Preparation of mitochondria and submitochondrial particles. Most experiments were carried out with mitochondria isolated by the method of Lambowitz et al. (12). Where specified in the text, the mitochondria were further purified by sucrose gradient centrifugation (16). For the preparation of submitochondrial particles, mitochondria (60 mg of protein) were suspended in 2.0 ml of 10 mM tris(hydroxymethyl)aminomethane (Tris)-SO4 (Sigma), pH 7.45. The suspension was disrupted by sonic treatment for 4 min with a Heat System W200 R cell disrupter equipped with a special microtip (power setting, 5; 40% duty cycle giving 1.6 min of total sonic treatment time), and submitochondrial particles were collected by the method of Tzagoloff (34). Enzyme assays. ATPase activity was assayed by three alternative procedures, as follows. (i) The liberation of inorganic phosphate was determined in 1.0 ml of reaction mixture containing 5 mM Na2ATP, 5 mM MgCl2, 50 mM Tris-glycylglycine buffer (pH 8.25), and an ATP regenerating system (5 mM phosphoenolypyruvate [PEP]-50 ug of pyruvate kinase [Sigma type II kinase]). The reaction was started by the addition of 15 to 100 yig of mitochondrial protein. After 5 or 10 min, the assay was terminated by the addition of 200 id of 55% trichloroacetic acid, and the tubes were centrifuged at 2,000 x g for 5 min. A portion of the supernatant fraction was then assayed for inorganic phosphate by the method of Dryer et al. (4).

585

(ii) Alternatively, ATPase activity was measured spectrophotometrically in a coupled enzyme assay system. In this case, the reaction mixture contained 5 mM Na2ATP, 5 mM MgCl2, 50 mM Tris-glycylglycine buffer (pH 8.25), ATP regenerating system (as above), 0.2 mM reduced nicotinamide adenine dinucleotide (NADH), and 50 ,ug of lactic dehydrogenase, in a total volume of 3.0 ml. The reaction was started by the addition of mitochondria, and the decrease in absorbance at 340 nm (due to the oxidation of NADH) was monitored with a Zeiss PMQ II spectrophotometer equipped with a Kipp and Zonen BDll lin-log recorder. (iii) Finally, ATPase activity was measured by the decrease in pH that accompanies the hydrolysis of ATP. The reaction mixture consisted of 0.1 mM Na2ATP, 0.1 mM MgCl2, and 1.0 mM tricine buffer (pH 8.25), in a 2.3-ml water-jacketed vessel. The reaction was started by the addition of mitochondria (1 to 3 id of a suspension containing 60 mg of protein per ml), and the rate of change of pH was measured with a Radiometer 25 pH meter attached to a Kipp and Zonen recorder. The total change in pH did not exceed 0.01 pH unit. All ATPase assays were carried out at 300C unless otherwise specified. Phosphatase activity was determinedwith5mMp-nitrophenylphosphateas substrate in the presence of 5 mM MgCl2 and 50 mM Trisglycylglycine buffer, pH 6.9 to 9.25. In some experiments, the liberation of inorganic phosphate was assayed by the method of Dryer et al. (4); in others, the appearance of p-nitrophenol was monitored spectrophotometrically, by adjusting the solution to an alkaline pH and measuring the absorbance at 410 nm. Protein assay. Protein was measured by the method of Lowry et al. (15) with bovine serum albumin as the standard. Reagents. Nucleoside triphosphates,p-nitrophenyl phosphate, PEP, NADH, pyruvate kinase, lactic dehydrogenase, tricine, glycylglycine, oligomycin, DCCD, adenylyl imidodiphosphate (AMP-PNP), carbonyl cyanide m-chlorophenylhydrazone, and chloramphenicol were obtained from Sigma Chemical Co.

RESULTS Effect of pH on ATPase activity. Figure

1A shows the ATPase activity of Neurospora mitochondria over the pH range 6.9 to 9.5. In both wild type and poky, the major peak of activity occurred at pH 8.25, with a smaller and more variable peak between pH 7.25 and 7.50. In the middle of the pH range (from pH 7.25 to 8.75), the ATPase activity of both strains was nearly completely inhibited by oligomycin (25 ,ug/mg of protein [Fig. 1B]), as expected for mitochondrial ATPase. In the same range, phosphatase activity was extremely low (0.02 to 0.06 ,tmol/min- mg of protein, assayed with p-nitrophenyl phosphate as substrate). Above pH 8.75, oligomycin-resistant ATPase (Fig. 1B) and phosphatase increased sharply; both of these activities could be reduced by passage of the mitochondria through a continuous sucrose gra-

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Because sensitivity to oligomycin can provide information about the association of mitochondrial ATPase with the inner membrane, it was important to compare the effects of a range of oligomycin concentrations on wild-type and poky mitochondria. The results of an experiment of this kind are shown in Fig. 2A. The ATPase activity in poky proved, if anything, to have a slightly increased sensitivity to oligomycin: halfmaximal inhibition occurred at 0.5 ,ug/mg of protein in poky compared with 1.0 ,ug/mg of protein in the wild type. Overall, 92 to 95% of the total ATPase activity was inhibited in both strains by the highest oligomycin concentration in this experiment (50 ,ug/mg of protein); in other experiments, the remaining 5 to 8% of activity was not abolished by concentrations as high as 200 ,ug/mg of protein. Two other mitochondrial ATPase inhibitors were tested (Fig. 2B): DCCD, which, like oligomycin, inhibits only the membrane-bound form of the enzyme (25), and AMP-PNP, which competes with ATP and inhibits both the soluble and membrane-bound forms (21). In both cases,

o _ >~~~~~~t

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90

70

pH

FIG. 1. Effect of pH

75

80

85

9.0

pH

on

the ATPase activity of

wild-type (wt) and poky mitochondria. (A), Control; (B) activity in the presence of oligomycin (25 pg/mg ofprotein). The liberation of Pi was determnined in a reaction mixture containing 5 mM Mg-ATP, 50 mM Tris-glycylglycine buffer (adjusted to the appropriate pH), and an ATP regenerating system (phosphoenolpyruvate plus pyruvate kinase).

dient, but because all further work was carried out at pH 8.25 (the optimum for the mitochondrial ATPase), conditions under which contamination by oligomycin-resistant ATPase and by phosphatase was less than 10%, the sucrose gradient step was normally omitted. Addition of 3 ,uM carbonyl cyanide m-chlorophenyl hydrazone did not stimulate ATPase activity at pH 8.25, presumably because the mitochondria were already fully uncoupled. One unexpected aspect of the results shown in Fig. 1 was the substantial amount of oligomycin-sensitive ATPase activity present in poky mitochondria. In this particular experiment, the specific activity of poky was 77% greater than that of wild type (2.1 and 1.3 umol/min- mg of protein, respectively). In other experiments, the specific activity averaged 1.93 ± 0.03 ,umol/min * mg of protein in poky (mean ± standard error of the mean for 10 determinations) and 1.33 ± 0.07 ,umol/min mg of protein in wild type (for 13 determinations). For convenience, the strain of poky used for these experiments carried the nuclear suppressor f, which increases the growth rate without restoring the wild-type cytochrome composition (18). To check the possibility that the fsuppressor mutation might be responsible for the surprising amount of oligomycin-sensitive ATPase activity seen inpoky, mitochondria were isolated from 21-h cells of an f-minus strain of poky and proved to have essentially the same characteristic: an ATPase activity at pH 8.25 of 2.84 ,tmol/min. mg of protein (even higher than that usually observed in f-plus poky), which was 85% sensitive to oligomycin (25 ,ug/mg of protein). All subsequent experiments were carried out with poky containing f. Effect of inhibitors of ATPase activity.

J. BACTERIOL.

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FIG. 2. Effect of oligomycin (A) and DCCD and AMP-PNP (B) on the ATPase activity of wild-type (wt) and poky mitochondria. ATPase activity was assayed at pH 8.25 as described in the legend to Fig. 1. Oligomycin and DCCD were added from ethanolic stock solutions, with the final ethanol concentration kept below 0.5%. (At higher oligomycin concentrations, 92 to 95% of the total activity was inhibited.) AMP-PNP was added from an aqueous stock solution.

587

MITOCHONDRIAL ATPase OF NEUROSPORA CRASSA

VOL. 133, 1978

half-maximal inhibition occurred at the same concentration in the wild type and poky (8 ,ug/mg of protein for DCCD and 6,uM for AMPPNP). Measurement of Km. ATPase activity was determined for both wild-type and poky mitochondria over a range of ATP concentrations, from 0.08 to 7.5 mM. Figure 3 is an Eadie-Hofstee plot of the data from one such experiment. The two preparations had very similar Km values (0.33 mM for the wild type and 0.29 mM for poky), but, as expected from results reported above, different maximal activities (1.2 pmol/min- mg of protein for the wild type and 2.0 muml/min. mg of protein for poky). In this particular experiment, ATPase activity was assayed as the liberation of inorganic phosphate, but essentially the same Km values (between 0.1 and 0.3 mM) were obtained by the pH or spectrophotometric assays. Substrate specificity. The ability of other nucleoside triphosphates to substitute for ATP is shown in Table 1. Generally, the wild-type and poky enzymes were very similar in both cases; ATP was the best substrate: GTP and ITP were hydrolyzed almost as rapidly, and UTP, CTP, and TTP were poor substrates. Effect of salts and of divalent cations. The effect of increasing salt concentrations in the presence and absence of Mg2e is shown in Fig. 4 for both wild-type and poky ATPases. To minimize the starting salt concentration in this experiment, the reaction was carried out in 1 mM tricine buffer (pH 8.25) at an ATP concentration of 0.1 mM, and ATP hydrolysis was measured by the pH assay. In the absence of Mg2e, ATPase activity was very low and was

TABLE 1. Substrate specifici Activity %

Nucleoside triphosphate

Wild type

poky

ATP 100 100 GTP 96 77 ITP 89 78 UTP 41 11 CTP 25 0 TTP 8 10 a Activity was assayed by the liberation of Pi in a reaction mixture consisting of 50 mM Tris-glycylglycine (pH 8.25) and 0.1 to 10 mM magnesium-nucleoside triphosphate. For each nucleoside triphosphate and each strain, the maximal activity was calculated by a computer fit of the data to the Michaelis-Menten equation. Activities are reported as the percentage of activity with ATP as substrate. The control rates with ATP were 1.0 ytmol/min mg of protein for the wild type and 1.7 ,umol/min mg of protein for poky.

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FIG. 4. Effect of NaCi and KCI onATPase activity ofpoky (A) and wild-type (wt) (B) mitochondria. ATPase activity was assayed by measuring the decrease in pH that accompanies the hydrolysis of ATP, as described in the text. The reaction mixture consisted of 0.1 mM Na2ATP, 0 or 0.1 mM MgCl2, and 1.0 mM tricine buffer (pH 8.25), plus 0 to 75 mM KCI or NaCI. Low activities reflect the fact that the assay was performed at 0.1 mM Na2ATP.

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FIG. 3. Dependence of ATPase activity on MgATP concentration: an Eadie-Hofstee plot of the data for wild-type (wt) and poky mitochondria. ATPase activity was assayed at pH 825 as described in the kgend to Fig. 1. The data were fitted to straight lines by the method of kast squares; correlation coefficients were greater than 0.9.

not increased upon addition of KCI or NaCl (up to 75 mM). In the presence of Mg2+, base-line activity was higher, and the addition of KCI or NaCl produced a further stimulation of threeto fivefold, with a half-maximal effect at approximately 8 mM salt both in the wild type and poky. Simultaneous addition of KCI and NaCl gave no evidence of synergism. When the buffer concentration was increased to 50 mM, the salt

effect was no longer seen. The Mg2e requirement of ATPase, which is shown in Fig. 4, was explored further by testing the ability of other divalent cations to substitute for Mg2+ (Table 2). In both wild-type and poky mitochondria, the same order of effectiveness was observed, with Mn2+ producing an even greater stimulation than Mg2+, Co2+ giving some

588

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MAINZER AND SLAYMAN

TABLE 2. Effects of divalent cations on the ATPase activity of wild-type and poky mitochondriaa Activity (%) Divalent cation

Wild type

poky

Mg2+ Mn2+ co2+

100 144

100 220

56

84

Ca2+ Cu2+ Fe2+

8

12 7 6

0

0 11 2 None a ATPase activity was assayed by measuring the release of H+ ions that accompanies the hydrolysis of ATP, as described in the text. The reaction mixture consisted of 0.1 mM divalent cation, 0.1 mM ATP, and 1.0 mM tricine buffer (pH 8.25); other experiments (data not shown) confirmed that maximal activity was obtained at a 1:1 ratio of divalent cation to ATP. Activities are reported as the percentage of activity with Mg20. The control rates with Mg2+ were 0.57 umol/min mg of protein for the wild type and 0.86 ,umol/min mg of protein for poky (the low rates reflecting the fact that the assay was carried out at 0.1 mM ATP).

activity, and Ca2+, Cu2+, and Fe2+ serving as very poor substitutes. Measurement of activation energy. A very sensitive way to detect abnormalities in a membrane-bound enzyme complex is to analyze enzyme activity as a function of temperature. We therefore determined the ATPase activities of wild-type and poky mitochondria over a range of temperatures, from 30 to 600C. When freshly isolated mitochondria were compared, three small but significant differences were observed, as follows. (i) Total ATPase activity and oligomycin-sensitive ATPase activity of poky mitochondria were higher than that of wild-type mitochondria at all temperatures tested, although oligomycin-insensitive activity was the same for the two strains. (ii) Maximal ATPase activity was observed at 550C in the wild type and at 600C (or above) in poky. (iii) Arrhenius plots revealed a 30% lower activation energy for the poky ATPase (33.7 kJ/mol) than for the wild-type ATPase (47.2 kJ/mol) (Fig. 5). These differences persisted in mitochondria that had been frozen in acetone-Dry Ice and thawed 3 h later, but diminished when the mitochondria were stored at -70°C for one week; in the latter case, maximal activity in both strains was observed at 550C, and the activation energies were 42.1 kJ/mol for poky and 37.9 kJ/mol for wild type. Retention of ATPase activity in submitochondrial particles. The results reported up to this point indicate that poky, in spite of its known defect in mitochondrial protein synthesis,

possesses an elevated specific activity of mitochondrial ATPase with only minor differences in kinetic properties and in activation energy. Furthermore, the fact that the poky ATPase is oligomycin sensitive implies that it is bound in a relatively normal way to the mitochondrial membrane. A second way to investigate binding is by the preparation of submitochondrial particles. In yeast, for example, disruption by sonic treatment leaves the wild-type mitochondrial ATPase in the particulate fraction but releases the ATPase of petite mutants into the supernatant fraction (28). To examine this point in Neurospora, mitochondria were prepared from the wild-type strain and from poky, disrupted by sonic treatment for 1.6 min, and centrifuged, and the various fractions were assayed for ATPase activity (Table 3). In both strains, oligomycin-sensitive ATPase activity was recovered in good yield in the particulate fraction (128% in the wild type and 99% in poky), indicating firm binding of the ATPase to the mitochondrial membrane. Response of ATPase activity to incubation in the cold. One well-established property of mitochondrial ATPase preparations from other organisms is their complex response to cold incubation. The complete oligomycin-sensitive ATPase complex, including the membrane subunits, is cold stable, but solubilized Fl ATPase (which is insensitive to oligomycin) loses its activity rapidly when incubated at 00C (24). 10

r

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300

310

I temperature (absolute)

320 X

330

105

FIG. 5. Arrhenius plot of the ATPase activity of wild-type (X) and poky (0) mitochondria. Activity was measured in the presence and absence of oligomycin (25 pg/mg of protein) at pH 8.25 and at temperatures of 30 to 60°C. Linear (increasing) portions were fitted by the method of least squares; correlation coefficients were greater than 0.9.

TABLE 3. ATPase activity in submitochondrial particlee Sp act

Fraction

Total units

(pmol/min)

(Qmol/ minm mg of protein) Plus oliControlgocn gomycin

Wild type Mitochondria Particulate fraction Supernatant fraction

120 145

1.19 2.38

0.12 0.21

17

0.49

0.14

86.5 85.4

2.22 3.06

0.48 0.73

0.9

0.06

0.07

poky Mitochondria Particulate fraction Supernatant fraction

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MITOCHONDRIAL ATPase OF NEUROSPORA CRASSA

VOL. 133, 1978

aMitochondria (101 mg in the case of the wild type and 39 mg in the case of poky) were suspended in 10 mM Tris-sulfate (pH 7.45) and disrupted by sonic treatment as described in the text. Between 36 and 46% of the total mitochondrial protein was usually released by sonic treatment. The suspension was centrifuged at 110,000 x g for 60 min. The resulting particulate and supernatant fractions were assayed for ATPase activity at pH 8.25 in the presence of 5 mM MgCl and 5 mM ATP, and in the presence and absence of oligomycin (13 to 40 ,g/mg of protein), as described in the legend to Fig. 1. Protein recovery was 101% for poky and 95% for the wild type.

sitive fraction remained constant for the entire 4-h period of the experiment, regardless of whether the mitochondria were kept at room temperature (Fig. 6, upper left) or at 0°C (Fig. 6, lower left). Warm-prepared poky mitochondria also exhibited an unusually high ATPase activity (4.0 ,umol/min mg of protein, compared with values of 1.93 + 0.03 umol/min mg of protein seen with cold-prepared poky mitochondria). The response during the 4-h incubation period was quite different in poky, however. When poky mitochondria were incubated at room temperature, total ATPase activity remained relatively constant, but there was a steady increase in the fraction insensitive to oligomycin (Fig. 6, upper right). It was equally striking that when poky mitochondria were transferred to 00C, there was a pronounced loss of total ATPase activity over the 4-h incubation period until, by the end of the incubation, activity had declined almost to the range characteristic of cold-prepared poky mitochrondria. It appears, therefore, that in addition to the large amount of relatively normal mitochondrial ATPase activity in poky, there is a further portion that is decidedly abnormal; it is cold labile and at room temperature gradually becomes insensitive to oligomycin during prolonged incubation. Effect of growth in the presence of chloramphemiol. As an independent check on the role of mitochondrial protein synthesis in the production of the Neurospora mitochondrial ATPase, cells were grown in the presence of

Cold stability can therefore be used as a third Poky Wild type criterion of normal attachment of Fl to the worm worin worm-_ worm mitochondrial membrane. All ofthe experiments 4.0 mitochondrial reported above for Neurospora yttal ctivity ATPase were carried out with mitochondria that .'l 3.0 octi,ity ,otol that 0 conditions had been prepared at to 40C, 2.0 X X x would completely inactivate Fl ATPase. It is clear, then, that the elevated mitochondrial oliOomycin ligoemycin inz nsitive insensitive /f ATPase activities seen in poky are norinal in E their cold stability. We thought it useful, howcold worm -_ cold ever, to see whether additional cold-labile ATP- :1 4.0 GI001 octivity ase activity might exist in wild-type and espe'0 4. cially in poky mitochondria. For this purpose, t0t01 octiolty mitochondria from both strains were prepared at room temperature, divided into two portions, and kept for 4 h at 25 and 00C, respectively, 0) 1.0 X,n,sen.sXitiVve oligomycin i l with periodic assays of ATPase activity (Fig. 6). 180 240 120 240 120 lSO In the wild-type mitochondria prepared at room Time ( min ) Time (min) temperature, total ATPase activity was some6. ATPase activity of wild-type (x) and poky what higher than usual (1.9 ,umol/min mg of (0)FIG. mitochondria prepared at room temperature and 0.07 protein, compared with 1.33 + 4 h either at room temperature (warm a for kept ,umol/min mg of protein for cold-prepared mi- warm) or at OC (warm cold). Activity was assayed tochondria); it was more than 90% inhibited by at pH 8.25 as described in the legend to Fig. 1, in oligomycin, and both the major oligomycin-sen- the presence and absence of oligomycin (25 pg/mg of sitive fraction and the minor oligomycin-insen- protein). -

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MAINZER AND SLAYMAN

chloramphenicol (4 mg/ml). Previous experiments have shown that such cells, like poky, contain greatly reduced amounts of cytochromes aa3 and b (14). Table 4 is a summar of the ATPase activities of cold-prepared mitochondria from chloramphenicol-grown wild type, poky, and (for comparison) chloramphenicolgrown yeast. In both strains of Neurospora, growth in the presence of chloramphenicol did not significantly alter the amount of cold-stable ATPase activity (Table 4). Furthermore, the ATPase remained oligomycin sensitive, although high concentrations of oligomycin (100 rig/ml of protein) were required to give complete inhibition. (The data in Table 4 show partial inhibition with 30 to 59 Lg of oligomycin per mg of protein.) By comparison, mitochondria isolated from chloramphenicol-grown yeast contained greatly reduced amounts of cold-stable ATPase that was only 30% inhibited by oligomycin. Thus, there appears to be a significant difference between Neurospora and yeast with respect to the role of mitochondrial protein synthesis.

DISCUSSION Properties of the Neurospora mitochondrial ATPase. The results reported in this paTALE 4. Mitochondrial ATPase activity in

chloramphenicol-grown cells Sp act (pmol/min mg of protein) Mitochondria Control

Plus ol-

gomycin

Wild-type Neurospora Normal Chloramphenicol grown

1.20 1.80

0.14 0.60

poky Neurospora Normal Chloramphenicol grown

1.93 1.65

0.18 0.79

Yeast Normal 1.30 0.25 0.35 Chloramphenicol grown 0.25 a Mitochondria were isolated as described in the text from wild-type and poky Neurospora grown in the presence of chloramphenicol (4 mg/ml). In addition, mitochondria were isolated by the method of Duell et al. (5) from yeast (Saccharomyces cerevisiae) grown for 17 h in 0.8% glucose-1% yeast extract-1% peptone-40 mg of adenine per liter-4 mg of chloramphenicol per ml. ATPase activity was assayed at pH 8.25 as described in the legend to Fig. 1 in the presence of 5 mM ATP and 5 mM MgCl, and in the presence and absence of oligomycin (45 jig/mg of protein for wild-type Neurospora, 31 jig/mg of protein for poky, and 59 ,g/mg of protein for yeast).

per demonstrate that Neurospora mitochondria contain substantial membrane-bound ATPase activity, as would be expected from the fact that Neurospora is an obligately aerobic fungus that produces most of its ATP by oxidative phosphorylation (33). Generally, the Neurospora enzyme is similar to the ATPase isolated from yeast and mammalian mitochondria. All are sensitive to the inhibitors oligomycin, DCCD, and AMPPNP, and all have Km values for Mg-ATP in the range 0.1 to 0.3 mM (1-3, 20, 27, 29, 37). There are some minor kinetic differences. The Neurospora ATPase has a pH optimum between 8.0 and 8.5, like beef heart (37) and rat liver (3) but unlike yeast (2, 27), for which maximal activity is seen above pH 9.0. However, in its nucleotide specificity and divalent cation requirements, the Neurospora ATPase is closer to yeast than to the mamimalia enzymes. Yeast and Neurospora hydrolyze ATP, GTP, and ITP at nearly identical rates, whereas rat liver and beef heart show a pronounced specificity for ATP (3, 37). Also, the yeast and Neurospora enzymes prefer Mn2" to Mg2e (2); mammalian mitochondrial ATPases prefer Mg2+, although they do function in the presence of Mn2+, Co2+, or Ca2` (5). Comparison of wild type and pohy. The most significant outcome of this study is the finding that the mitochondrial ATPase of poky, when characterized in the membrane-bound state in cold-prepared mitochondria, is nearly indistinguishable from that of the wild type. Thus, in spite of a clear-cut defect in mitochondrial protein synthesis that grossly impairs the production of cytochromes aa3 and b (12, 31, 41, 42, 44, 45), poky makes a substantial amount of mitochondrial ATPase that is cold stable, is sensitive to oligomycin, and has normal kinetic properties. Whether the poky ATPase functions normally in oxidative phosphorylation is not yet clear. Cytochrome-linked phosphorylation in isolated mitochondria is only about one-half as efficient in poky as in the wild type. For example, in the study of Lambowitz et al. (14), wild-type mitochondria respiring on succinate gave ratios of phosphorus to oxygen of 1.3, whereas poky mitochondria respiring on succinate in the presence of salicyl hydroxamic acid (to block the alternate oxidase) gave ratios of phosphorus to oxygen of about 0.75. This difference could reflect an abnormality in the functioning of the ATPase complex itself, but it could arise equally well from lesions elsewhere in the inner membrane. (For example, isolated poky mitochondria have been shown to be more permeable to small ions than are isolated wild-type mitochondria [7], and according to one leading theory of oxidative phosphorylation [19], increased "leakiness" of the

VOiL. 133, 1978

MITOCHONDRIAL ATPase OF NEUROSPORA CRASSA

inner membrane should dissipate the potential generated by the redox chain, thereby decreasing the efficiency of phosphorylation.) The implications of these findings for the biogenesis of the mitochondrial ATPase complex remain to be worked out. The relative normality of the pohy ATPase in cold-prepared mitochondria and the fact that very similr results were obtained with cold-prepared mitochondria from chloramphenicol-grown celLs indicate that products of mitochondrial protein synthesis play only a limited role in the functioning of the Neurospora mitochondrial ATPase, in contrast with their clear-cut role in attaching Fl to the membrane and conferring oligomycin sensitivity in yeast (38). In pursuing this question, it will be important to purif an active membrane-bound ATPase from Neurospora, as well as an active Fl ATPase, and to compare the subunits present in these complexes in wild type,poky, and chloramphenicol-grown wild type. In this way, it should be possible to clarify the role of the mitochondrially synthesized polypeptides described by Jackl and Sebald (6) and to understand the basis for oligomycin sensitivity. A second significant result of this study is the finding that, in addition to its cold-stable, oligomycin-sensitive ATPase, poky contains a significant amount of ATPase that is associated with the mitochondria in an oligomycin-sensitive manner but is cold labile. Taken together, the total specific activity of the two forms ofATPase in poky (4.0 ,umol/min- mg of protein in warmprepared mitochondria) exceeds the specific activity of the ATPase of wild-type mitochondria (1.9 ,umol/min- mg of protein), suggesting that mitochondrial ATPase may belong to the group of mitochondrial proteins (including cytochrome c and the alternate terminal oxidase [13]) that are produced in elevated amounts in poky. Two alternative explanations of the specific activity data can be ruled out on the basis of existing evidence, as follows. (i) If poky mitochondria were abnormally fragile, damage during the isolation procedure might lead to loss of soluble matrix protein and consequently to enrichment of ATPase attached to the inner membrane. This explanation is unlikely, however, because mitochondria from the wild type and from poky display identical osmotic behavior in sucrose solutions and have the same sucrose-impermeable space (7). (ii) If poky cells contained a normal number of ATPase complexes inserted into a decreased number of mitochondria, the result would be an increase in the specific activity of the ATPase. This explanation, too, seems unlikely, because the standard preparation procedure (treatment of cells with snail enzyme to weaken their cell walls, followed by gentle ho-

591

mogenization) routinely releases 1.5- to 2-fold more mitochondrial protein from poky than from the wild-type strain (S. E. Mainzer and K. E. Allen, unpublished data). The possibility remains that the increased specific activity of the poky ATPase reflects an increased turnover number resulting, for example, from abnormal binding of an inhibitory subunit or from abnormal association of the ATPase with the mitochondrial membrane. Whether this is the case or whether ATPase complexes are truly produced in elevated amounts in poky should become evident from purification of the ATPase and from studies of its subunit composition. ACKNOWLEDGMENTS We are grateful to Kenneth Allen for expert technical assistance. This work was supported by a Public Health Service traineeship (grant GM-02299) awarded to S.E.M. and by research grant GM-15761, both from the National Institute of General Medical Sciences.

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