Mitochondrial biogenesis during fungal spore germination

ARCHIVES

OF BIOCHEMISTRY

Mitochondrial

AND

BIOPHYSICS

182,

Biogenesis

Development

273-281

during

Department

of

Fungal

of Cytochrome

ROBERT

(1977)

c Oxidase

Germination

Activity’

BRAMBL

Plant Pathology, The University of Minnesota, Received

Spore

February

Saint Paul, .Minnesota 55108

4, 1977

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibiters of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-ccysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de nouo upon germination.

The organization of new mitochondria and the development of mitochondrial function require coordinated activity of both the nuclear and mitochondrial genetic systems. An examination of mitochondrial biogenesis during the germination of metabolically quiescent, dormant fungal spores into rapidly growing cells may provide useful insight into certain mechanisms of mitochondrial respiratory system assembly, as well as an increased understanding of the organism’s biochemical and developmental transition from dormancy to active growth. Eventually it should be possible to determine the required order or sequence of synthesis of 1 Paper No. 9703, nesota Agricultural Paul, Minnesota.

Scientific Journal Series, Experiment Station,

nuclear and mitochondrial gene products, while simultaneously establishing which components of the mitochondrial respiratory system are preserved in the dormant spore for function during germination (in the absence of new synthesis) and whether any of these components must be synthesized de novo before the respiratory system may function. During germination of conidiospores of Botryodiplodia theobromae, the initiation of function of cytoplasmic and mitochondrial ribosomes is temporally separated; the cytoplasmic ribosomes begin translation of a latent messenger RNA immediately upon introduction of the spores into a liquid medium (1, 2>, whereas the mitochondrial ribosomes become active about 90 min later (3). Within 30 min of inocula-

MinSaint 273

Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003-9861

274

ROBERT

tion, the spores begin an accelerating oxygen uptake which is abolished by both cyanide and cycloheximide but is insensitive initially (for 150 min) to inhibitors of the mitochondrial genetic system (2). Although the mitochondria of these dormant spores contain cytochrome c and one of the two cytochromes b of the germinated spores, they do not contain cytochrome a or heme a (4). However, after 150 min of incubation, the spores appear to initiate cytochrome heme synthesis, develop the normal complement of spectroscopically detectable cytochromes, and begin a second, accelerated phase of oxygen consumption. Cytochrome c oxidase (EC 1.9.3.1, ferrocytochrome c:oxygen oxidoreductase) is a respiratory enzyme whose synthesis and assembly are paradigmatic of the entire mitochondrial respiratory membrane [cf. Ref. (5) for a review]. Our previous results (4) indicated an absence of cytochrome a in mitochondria of dormant B. theobromae spores; the present study was undertaken to examine the activity of cytochrome c oxidase during spore germination and to establish some of the conditions required for elaboration of the enzyme activity. MATERIALS

AND METHODS

Techniques for production and germination of the spores and for preparation of the mitochondrial fraction have been described in detail previously (2). However, in the present study, the incubated spores were harvested and collected into glass tubes at each of the indicated times, and they were rapidly frozen in a solid CO,-acetone bath. The spores were stored at -20°C (usually about 18 h) until mitochondria were extracted; experiments showed that freezing of the spores in this way had no effect upon the extractable cytochrome c oxidase activity at any stage of germination when compared with extraction of freshly harvested spores. These spore samples (about 200 mg fresh weight each) were disrupted with a Braun MSK homogenizer equipped with a CO+ooled microchamber accessory containing 11.25 g of l.O-mm glass beads and 4.2 ml of extraction buffer, optimum proportions for obtaining >99% spore disruption after 30 s at 4000 rpm. The extraction buffer contained mannitol (0.25 M), EDTA (1.0 mM), and Tris (20 rnr.0 adjusted to pH 7.4 with H,SO,. The mitochondrial fraction, suspended in extraction buffer and positioned in an ice bath, was subjected to two 30-s bursts of sonic irradiation (with a

BRAMBL 30-s pause) from the 0.75-in. horn of a Braun-Sonic 1510 sonic generator operated at 100 W. After centrifugation of this suspension at 105,OOOg (g values are g,,) for 30 min, the resulting supernatant fluid was discarded and the pelleted material was suspended in 1 to 2 ml of the extraction buffer (above) which contained T&on X-100 (2%, v/v) and glycerol (20%, v/v). The resuspended fraction was centrifuged at 12,lOOg for 10 min; the resulting supernatant fluid, containing the cytochrome c oxidase activity was dispensed into small vials and stored at -20°C until assay (usually 12 to 36 h) with no apparent loss of activity when compared with the results of assays of freshly prepared material. The ferrocytochrome c was prepared by dissolving horse heart cytochrome c (Type III, Sigma Chemical Co.) in 0.01 M (anion concentration) KPO, buffer (pH 7.4) to a concentration of 10 mg/ml. Solid sodium ascorbate was added to this solution in excess (the reduction was followed spectrophotometrically), and the ascorbate was removed chromatographically by elution of the solution through Sephadex G-25. The ferrocytochrome c was dispensed into small vials, flooded with nitrogen gas, and stored at -20°C until used. The cytochrome c oxidase assay reaction mixture, in a total volume of 1.0 ml, contained 0.1 M KPO, buffer (pH 5.91, 1% (v/v) Tween 20, and 15 WM ferrocytochrome c. Only cytochrome c that was reduced 95% or more was used, as determined by 550/565-nm absorbance ratios (6). Small volumes (10 to 100 ~1) of the fraction to be assayed for cytochrome c oxidase activity were added to this reaction mixture and the initial linear change in absorbance at 550 nm was recorded with a Cary 118C spectrophotometer. At the conclusion of the reaction, a drop of a freshly prepared, saturated solution of K,Fe(CN), was added to the reaction mixture to oxidize the cytochrome c completely. The specific activities of the enzyme fractions were calculated from changes in absorbance and expressed as micromoles of cytochrome c oxidized per minute per milligram of solubilized protein. Control assays were performed on all samples by the addition of NaCN to the reaction mixture to a final concentration of 1 mM prior to the addition of sample enzyme protein. Protein concentrations were estimated by the method of Lowry et al. (7), using bovine serum albumin as a standard. Coproporphyrin III was prepared (immediately before use) from the commercial tetramethyl ester (Type III-H, Sigma Chemical Co.) by hydrolysis with 6 N HCl at 23°C for 18 h, the HCl was removed by vacuum evaporation. Sigma or Calbiochem supplied all inhibitors and analogs except cerulenin, which was a generous gift of Prof. S. Omura. All the experiments described in this report were performed two or more times, and the examples cited represent typical results.

CYTOCHROME

c OXJDASE

RESULTS

The 240-min germination sequence of the B. theobromae spores included a preemergence phase (about 150 min) during which none of the spores showed germ tubes and an emergence phase (about 90 min) during which 90 to 95% of the spores developed elongating germ tubes under previously specified (2) eonditions of incubation in nutrient medium. The dry weight of the spores did not change during the first 240 min of germination, but after 300 min the dry weight began to increase with a mass doubling time of 4 h. At the outset of this study, several conditions for enzyme fraction preparation and assay were tested and compared to obtain the maximum measurable specific activity of cytochrome c oxidase in extracts from germinated spore mitochondria. Maximum activity was obtained when the sonicated mitochondria were suspended in a medium containing Triton X-100 (2%); use of other detergents, such as sodium cholate (1%) or digitonin (l%), caused a substantial reduction in enzyme activity. Storage of the enzyme in Triton X-100 and glycerol (20%) at -20°C ensured stability; use of 50% glycerol, 10% Tween 80, or (NH&SO, (at 35% of saturation at O’C) reduced the activity, and storage of the enzyme fraction at -80 or -196°C in any of these solvents did not increase stability. Inclusion of 1% Tween 20 (but not 1% Tween 80) in the reaction mixture increased enzyme activity almost 50%. The solubilized cytochrome c oxidase activity was completely dependent upon exogenous ferrocytochrome c’, and the reaction was found to be pseudo-first order with respect to ferrocytochrome c concentration, as others also have observed (8-10). The K, of cytochrome c in the reaction was calculated from a double-reciprocal plot to be 2.2 x lo-” M, a value identical to that obtained with a similar enzyme fraction from yeast mitochondria (ll), but with different preparation and assay conditions. The pH curve for the reaction in KPO, buffer (100 mu) was rather sharp, with an optimum at pH 5.9, and the optimum PO, concentration (at pH 5.9) was judged to be 100 mM; in all subsequent experiments the enzyme assays were performed at these optima.

IN

FUNGAL

SPORES

275

The cytochrome c oxidase activity extracted from mitochondria of 240-min germinated spores was nearly abolished (>98%) by 1 IIIM NaCN, and, in each assay cited here, the small amount of cyanideinsensitive activity was subtracted from the total activity. The oxidation of ferrocytochrome c was not affected by the inclusion of catalase (288 units) in the reaction mixture. In Column A of Table I is shown the distribution of cytochrome c oxidase activity in the several centrifugation fractions obtained from 240-min germinated spores. In order to measure more accurately the enzyme activity in the fractions preceding mitochondrial disruption (steps l-4), it was necessary to carry an aliquot of each fraction through the sonication, highspeed centrifugation, and Triton X-lOO-solubilization steps (described in the methods section). The insoluble proteins, remaining after the final centrifugations, were not included in the reaction mixture, but the specific activity of each of these fractions is based upon the protein content of the entire cell fraction. This preparation of the cytochrome c oxidase fraction resulted in a ninefold enrichment of the enzyme specific activity over that found in the 5OOg soluble cell extract. It is evident that the majority of the enzyme activity is associated with a particulate mitochondrial fraction until the final solubilization step; however, a large amount of activity remained soluble at 27,000g in centrifugation step 2. It seems likely that this loss is due to disruption of mitochondria by the mechanical homogenizer; while I sought to obtain 100% spore disruption in this experiment, if the extent of spore breakage was reduced to 85%, the loss of cytochrome c oxidase from the mitochondria was reduced almost fivefold. In Column B of Table I it is shown that no cytochrome c oxidase activity appeared in the mitochondrial fraction prepared from the dormant spores. Assays of each of the spore fractions demonstrated that the enzyme activity was not inadvertently lost from the mitochondrial fraction in the preparation procedure. The spore disruption was complete, with 100% breakage, and the mitochondria prepared from these spores (fixed with glutaraldehyde and

276

ROBERT

BRAMBL

TABLE FRACTIONATION

Fractionation

OF PROTEIN

step”

1. First 500g Supernatan@ Precipitate 2. First 27,000g Supernatant Precipitate 3. Second 500g Supernatant Precipitate 4. Second 27,000g Supernatant Precipitate 5. 105,000g Supernatant Precipitate

AND CYTOCHROME

I

c OXIDASE ACTIVITY min) SPORES

Column A (germinated) Total protein (mg)

Specific

Total

294 128

0.130 0.074

220 62

Activity”

FROM

DORMANT

AND GERMINATED

(240

Column B (dormant) Total protein

Activitp

(mg)

Specific

Total

38.22 9.47

303 108

0 0

0.030 0

0.049 0.377

10.78 23.37

153 99

0 0

0 0

53 12

0.480 0.118

25.44 1.42

102 7

0 0

0.001 0.002

25 31

0.016 0.834

0.40 25.85

41 65

0 0

0.008 0

16 13

0.088 1.152

1.41 35 0 0 14.98 22 0 0 0 After disruption of 3.1 g of germinated spores and 3.0 g of dormant spores, as described in the text. b Cyanide-sensitive activity, micromoles of cytochrome c oxidized per minute per milligram of protein. e 0 activity 5 0.0003 ~mol/min/mg of protein. d Represents fractions combined from two serial washings and 5OOg precipitations.

stained with 0~0, for thin-s&ion observation in an electron microscope) were judged to be as intact morphologically as those prepared from germinated spores under the same conditions. In these dormant spore extracts, I was unable to detect any activity of cytochrome c oxidase over a range of protein concentrations up to 40 times that used in the assay mixtures of the germinated spore enzyme fraction. To test the possibility that an endogenous inhibitor of cytochrome c oxidase could cause the apparent absence of enzyme activity in the dormant spore extracts, a series of mixing experiments was conducted in which the active enzyme fraction from germinated spores was combined in the assay mixture with fractions from the dormant spores. A mitochondrial membrane extract from dormant spores, which was a counterpart of the active enzyme from the germinated spores, had no influence upon the germinated spore enzyme activity. Likewise, a complete mitochondrial fraction as well as the postmitochondrial supernatant fluid (intentionally kept as concentrated as possible) prepared

from the dormant spore mitochondria also did not inhibit the enzyme activity from the germinated spore preparations. These results demonstrate that the dormant spores do not contain a soluble inhibitor of cytochrome c oxidase. I sought to establish whether such an absence of cytochrome c oxidase might be characteristic of other dormant fungal spores under these conditions of assay. The mitochondrial fractions from viable, dry spores of three other phylogenetically diverse genera of fungi were extracted, and the enzyme fraction was prepared with methods identical to those used for B . theobromae. Dormant spore extracts of the following three species showed the enzyme spticific activities (micromoles of cytochrome c oxidized per minute per milligram of protein) indicated here in parentheses: Neurospora crassa conidiospores (1.7661, Rhizopus stolonifer sporangiospores (0.394), and Puccinia graminis f. sp. tritici uredospdres (0.059). These results demonstrate that some other dormant, fungal spores do contain significant quantities of cytochrome c oxidase (in con-

CYTOCHROME

c OXIDASE

trast to B. theobromue) and that our assay system is sufficiently sensitive to measure this enzymatic activity in other dormant spore extracts. Of particular interest in the present study was the measurement of the elaboration of cytochrome c oxidase activity during the germination of the B. theobromue spores. The pattern of extractable enzyme activity which occurs during spore germination is illustrated in Fig. 1A. It is notable that the spore mitochondria contained no cytochrome c oxidase activity until after 120 min of germination. At 150 min the enzyme activity began to rise rapidly and approached its maximum at about 240 min. The increase in enzyme activity was abolished if the spores were treated with cycloheximide (25 pg/ml) from the initiation of germination. If, however, the spores were germinated in the presence of chloramphenicol (3 mglml) or ethidium bromide (5 pg/ml), drugs which we previously have shown to inhibit mitochondrial ribosome function and mitochondrial DNA synthesis during germination of these spores (3, 12), the enzyme activity nevertheless began to increase after 120 min, and continued to increase rapidly, al-

IN

FUNGAL

277

SPORES

though the final level of activity attained was somewhat less than that of the control spores (Fig. 1A). If these spores are incubated in distilled water or distilled water containing Tween 80 (0.5%, v/v>, instead of the nutrient medium (which also contains Tween SO), several metabolic activities are begun which are also characteristic of spores incubated in the nutrient medium, but syntheses of RNA and DNA are not initiated (see Discussion). Under these conditions of incubation in water or Tween 80, in which only the stored nuclear messenger RNA is translated, I also found that the activity of cytochrome c oxidase increased during the first 240 min of incubation, although the kinetics of the increase were different for spores incubated in the two media (Fig. 1B). The enzyme activity of spores incubated in Tween 80 increased sharply, beginning at the same time as that of spores incubated in the nutrient medium, although the eventual level of cytochrome c oxidase activity attained was somewhat lower than that of the nutrient medium control and similar to that of spores germinated in chloramphenicol or ethidium bromide (Fig. lA>. In contrast, the development of enzyme activ16-

1.4 6t5

0

6--6-@--h-h 1 1 0 30

/ 60

Minutes

I 90

/ I 120

1 150

of Germinotmn

1 180

1 210

1 240

o-

B

,,,) o-:-:+-:-m/ 0 30

60

90

Mmutes

I20

150

/

/

,830

210

of Germmatton

FIG. 1. Kinetics of development of cytochrome c oxidase activity: (A), in germinating spores (0) and in spores germinating in the presence of chloramphenicol (A), ethidium bromide (A), or cerulenin (0); and (B), in germinating control spores (O), 1% Tween 80-incubated spores (O), and distilled water-incubated spores (m).

240

278

ROBERT

ity of spores incubated in distilled water was retarded, and by 240 min of incubation the amount of extractable activity was less than half that of Tween 80-incubated spores. Fewer than 10% of the spores incubated in Tween 80 had germinated by 240 min, while none of the spores in distilled water germinated. Cerulenin is an antibiotic which is reported to be a potent inhibitor of fatty acid biosynthesis (15). At a concentration of 50 pg/ml, this drug reduced B . theobromae spore germination by 95% at 240 min, and the development of cytochrome c oxidase activity was severely retarded in these inhibited spores (Fig. 1A). In Fig. 2 are shown the effects of addition of cycloheximide (25 pg/ml) to the spores at several times during germination upon the development of cytochrome c oxidase.activity. If the drug was present in the incubation medium at any time before 95 min, the elaboration of the enzyme activity was completely blocked; if cycloheximide was added at 100 min of germination, the mitochondria contained only a small amount of active enzyme. Addition of the

BRAMBL

II 0

I 30

I 60

Minutes

I 90

I 120

I 150

I IS0

I 210

I_ 240

of Germination

FIG. 3. Kinetics of cytochrome c oxidase development in spores treated with rcethionine (0) or S-2aminoethyl-L-cysteine (0) and in uninhibited germinating spores (0).

drug at 105 or 120 min permitted correspondingly greater amounts of the enzyme activity to be elaborated during the subsequent periods of the germination sequence. These results suggest that the obligatory time for synthesis of the proteins required for cytochrome c oxidase ac1.6 tivity occurs before about 100 min of germination, and after this point a sufficient quantity of proteins synthesized on cytoplasmic ribosomes is available for formation of a functional enzyme complex which is active in the in vitro assays. If one or more proteins required for development of cytochrome c oxidase activity are synthesized de nouo upon germination, as the experiments with cycloheximide inE 0.6dicate, the development of this enzyme ac:: tivity should be at least partially inhibited x 04” if the spores are incubated (from zero time) f in the presence of antagonistic amino acid g o-aanalogs. The results presented in Fig. 3 i _ 1 show that m-ethionine (200 pg/ml) .or S-2: o- - --.4 1 / I aminoethyl-L-cysteine (100 pg/ml), ana0 30 60 90 120 150 IS0 210 240 logs of methionine and lysine, respecMinutes of Germinotion tively, both caused significant depression FIG. 2. Kinetics of development of cytochrome c in the amounts of cytochrome c oxidase oxidase activity in germinating spores treated with extracted from the germinating cycloheximide at 95 min of incubation (O), 100 min activity spores. The lysine analog was particularly (A), 105 min (O), and 120 min (A). The enzyme both retarding the kinetics of development by untreated spores is represented by inhibitory, development of the enzyme activity and (0).

CYTOCHROME

c OXIDASE

limiting the amount of activity in the 240min incubated spores to about 25% of that in the untreated spores. Activity of this enzyme probably was not inhibited completely because of simultaneous or preferential use of endogenous methionine and lysine by the spores. The presence of either of these analogs reduced spore germination to about 2% of that of the untreated spores by 240 min. Coproporphyrin III is reported to be an intracellular inhibitor of de nouo synthesis of cytochrome c oxidase in anaerobic yeast cells whose inhibitory effect is lost upon aeration of the yeast culture (13). Although other experiments (described above) showed that the dormant B. theobromae spores contained no soluble inhibitor of the activity of cytochrome c oxidase, I wished to establish whether these spores contained a factor (such as coproporphyrin III) which blocked synthesis of proteins required for elaboration of the enzyme in germinating spores. Exogenous coproporphyrin III is maximally effective at a concentration of 5 WM in inhibiting accumulation of cytochrome c oxidase in yeast cells (13). I added several concentrations (up to 14 PM) of this porphyrin to spores at the initiation of germination and found no influence upon the subsequent elaboration of cytochrome c oxidase activity between 150 and 240 min of germination. Provided the spores are permeable to the porphyrin, these results indicate that coproporphyrin III does not have an effect in these spores like that which has been reported for cytochrome c oxidase of yeast cells. However, to establish whether some other soluble factor in the dormant spores could influence the synthesis of proteins required for cytochromk c oxidase activity, concentrated postmitochondrial supernatant fractions were prepared from dormant spores, from the parent pycnidia and mycelium, and from germinated spores. I found that the fraction from dormant spores (when added to the germination medium) reduced the spore germination to less than 7% of the control value while simultaneously reducing the amount of cytochrome c oxidase activity in the treated spores to about 30% of the control value

IN

FLJNGAL

SPORES

279

when added at any point of germination through the first 45 min. The postmitochondrial supernatant fractions from the parent pycnidia or from the germinated spores instead stimulated the rate of spore germination slightly and had no influence upon cytochrome c oxidase activity. Subsequent experiments have shown that this germination inhibitor is restricted to the spores alone, it is soluble in water and insoluble in ethyl acetate or methylene dichloride, and it probably has an indirect effect upon cytochrome c oxidase as a result of its activity elsewhere in the germinating spore. DISCUSSION

The experiments described in this report show that the dormant spores of Botryodiplodia theobromae do not contain a functional cytochrome c oxidase. This observation supports our earlier conclusion that the mitochondria of these spores do not contain cytochrome a or heme a (4). During spore germination, however, the enzyme activity increases rapidly after 150 min of spore incubation, the approximate time of new cytochrome synthesis (4) and the time at which a second, accelerated phase of oxygen uptake is begun (2). The elaboration of cytochrome c oxidase in these spores is abolished by cycloheximide, an inhibitor of cytoplasmic ribosomes, if this drug is added at or before 95 min of germination, but the development of this enzymatic activity is initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol and ethidium bromide. [Our previous studies (3, 12) have shown that the spores are permeable to these drugs and that they do inhibit mitochondrial ribosome function and DNA synthesis, respectively.1 The antagonistic amino acid analogs nL-ethionine and S-2aminoethyl+cysteine caused substantial reductions in development of cytochrome c oxidase in germinating spores. Provided that these analogs are inhibiting synthesis of functional proteins primarily, the results suggest that a portion of the cytochrome c oxidase enzyme or a protein required for its activity is synthesized de

280

ROBERT

nouo upon germination. When B. theobromae spores are incubated in distilled water or 1% Tween 80, the first phases of oxygen uptake and cycloheximide-sensitive protein synthesis are begun (2) in the absences of mitochondrial ribosome function (R. Brambl, unpublished data) and cellular nucleic acid synthesis (1, 2). The spores do not germinate in distilled water, and in Tween 80 (in the absence of nutrients) fewer than 10% of them germinate. This behavior in different incubation media provides a useful comparison to help establish the metabolic requirements for germination and indirectly to identify molecular components which are stored in the dormant spore and capable of function in the absence of new nuclear messenger RNA synthesis. It is significant, therefore, that in the present study cytochrome c oxidase activity was elaborated by spores incubated both in distilled water and in Tween 80; the retarded development of this enzyme in the distilled water-incubated spores and the near normal kinetics of development of the enzyme activity by spores incubated in Tween 80 may reflect a requirement (in addition to cytoplasmic protein synthesis) of membrane lipid for organization of the functional cytochrome c oxidase complex. This speculation is supported by experiments in the present study in which cerulenin, an inhibitor of fatty acid biosynthesis (15), severely reduced the rate and extent of spore germination and development of cytochrome c oxidase activity. ’ The absence of cytochrome c oxidase in dormant spores of B. theobromae may be relatively novel, since mitochondria from dormant spores of three other phylogenetitally diverse genera of fungi examined here contained readily detectable quantities of this enzyme. The dormant spores of B. theobromue appear not to contain soluble inhibitors of either cytochrome c oxidase function or synthesis, but an incidental observation made in this study suggests that the spores may contain an inhibitor of spore germination which indirectly reduces (but does not abolish) cytochrome c oxidase activity by 240 min of incubation. Cytochrome c oxidase of yeast and Neu-

BRAMBL

is a multiple-component enzyme whose subunits are derived from both the cytoplasmic and mitochondrial ribosomes and are encoded within nuclear and mitochondrial genes (5). Because function of cytoplasmic ribosomes (but not mitochondrial ribosomes) is required for oxygen uptake, germination, and development of a functional cytochrome c oxidase in the germinating spores of B. theobromae, it seems plausible that mitochondria of the dormant spores do not contain the enzyme subunits synthesized on cytoplasmic ribosomes but do contain the enzyme subunits synthesized on mitochondrial ribosomes. It is possible that the genetic information required for synthesis of the cytoplasmic subunits of this enzyme during germination may be stored in the latent nuclear messenger RNA of the dormant spores which we have shown to be translated early in germination in the absence of new mRNA synthesis (1). Translation of this messenger RNA early in germination may contribute the required cytoplasmic subunits of cytochrome c oxidase to the incomplete complex of mitochondrial subunits to permit the rapid elaboration of enzyme activity after 150 min. We have shown previously (3) that proteins synthesized on cytoplasmic ribosomes are incorporated into or associated with the mitochondria during the first 60 min of germination in the absence of mitochondrial ribosome function. While it is possible that the enzyme subunits from cytoplasmic ribosomes could be translated from new messenger RNA synthesized after 45 min of germination (14), the fact that the enzyme is elaborated in water-incubated spores [which are synthesizing no RNA or DNA (1, 2)l makes this possibility seem unlikely. Alternatively, it is also possible that the protein(s) synthesized on the cytoplasmic ribosomes are not enzyme subunits but, instead, are required for respiratory membrane assembly, and they affect the organization and function of this respiratory enzyme only indirectly. Either hypothesis, however, can be tested directly by an examination of synthesis of the individual cytochrome c oxidase subunits during spore germination. These experiments are now in progress. rosporu

CYTOCHROME

c OXIDASE

ACKNOWLEDGMENTS The author is grateful to B. Handschin for his expert assistance in this study. This research was supported in part by a Faculty Grant-in-Aid of Research from the University of Minnesota Graduate School and by NIH Research Grant GM-19398 from the National Institute of General Medical Sciences.

REFERENCES 1. BRAMBL, R. M., AND VAN EWEN, J. L. (1970) Arch. Biochem. Biophys. 137, 442-452. 2. BRAMBL, R. (1975) Biochim. Bzi~phys. Actu 396, 175-186. 3. BRAMBL, R., AND HANDSCHIN, B. (1976) Arch. Biochem. Biophys. 175, 606617. 4. BRAMBL, R., AND JOSEPHSON, M. (1977)5. Bacteriol. 129, 291-297. 5. SCHATZ, G., AND MASON, T. L. (1974)Annu. Rev. Biochem. 43, 51-87.

IN

FUNGAL

SPORES

281

6. PAUL, K.-G. (1947) Arch. B&hem. 12, 441-450. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951)J. Biol. Chem. 193, 265-275. 8. SMITH, L., AND CONRAD, H. (1956) Arch. Biothem. Biophys. 63, 403-413. 9. MINNEART, K. (1961) Biochim. Biophys. Actu 50, 23-34. 10. ERREDE, B., HAIGHT, G. P., JR., AND KAMEN, M. D. (1976) Proc. Nut. Acud. Sci. USA 73, 113117. 11. SEKUZU, I., MIZUSHIMA, H., HIROTA, S., YUBISLJI, T., MATSUMURA, Y., AND OKUNUKI, K. (1967) J. Biochem. (Tokyo) 62, 710-718. 12. DUNKLE, L. D., VAN ETTEN, J. L., AND BRAMBL, R. M. 1972. Arch. Mikrobiok 85, 225-232. 13. CHARALAMPOUS, F. C. (1974) J. Biol. Chem. 249, 1014-1021. 14. KNIGHT, R. H., AND VAN EVEN, J. L. (1976) J. Gen. Microbial. 95, 257-267. 15. OMURA, S. (1976) Bucteriol. Reu. 40, 681-697.