JOURNAL OF BACrERIOLOGY, Feb. 1976, p. 389-397Copyright 0 1976 American Society for Microbiology

Vol. 125, No. 2Printed in U.S.A.

Cytochrome Abnormalities and Cyanide-Resistant Respirationin Extranuclear Mutants of Aspergillus nidulans

GEOFFREY TURNER* AND ROBERT T. ROWLANDS

Department of Bacteriology, University of Bristol, Bristol BS8 lTD, United Kingdom

Received for publication 11 August 1975

The cytochrome spectra of two extranuclear mutants of Aspergillus nidulansand the double-mutant recombinant formed from them have been examinedboth at room temperature and at the temperature of liquid N2 and comparedwith those of the wild-type strain. The oligomycin-resistant, slow growing mutantcontained an increased amount of cytochrome c without any loss of cytochromesb and a,a8. The cold-sensitive mutant, apparently normal when grown at 37 C,showed an increased amount of cytochrome c and a partial loss of cytochromes band a,a3 when grown at 20 C. A combination of these effects was observed in thedouble-mutant recombinant. Cyanide-resistant respiration was present in bothmutant strains and in the recombinant at much higher levels than in thewild-type strain. In the oligomycin-resistant mutant, this was usually presenttogether with cyanide-sensitive respiration, whereas in the cold-sensitive mutantand recombinant grown at 20 C cyanide-resistance approached 100%. Inhibitorand growth yield studies indicated that the cyanide-resistant pathway was notused by the cold-sensitive mutant during growth at 20 C.

Extranuclear mutations affecting respirationhave been known for some time in Sac-charomyces cerevisiae (6) and Neurospora crassa(20), and, more recently, extranuclear mu-tants resistant to the mitochondrial adeno-sine 5'-triphosphatase inhibitor oligomycinhave been studied in S. cerevisiae (1, 7). Wehave previously reported isolation of the ex-tranuclear, oligomycin-resistant mutant (oliA1)in the filamentous ascomycete Aspergillusnidulans, which grows slower than the wild-typestrain on drug-free media (21), and preliminaryresults indicated an alteration to its cytochromespectrum (22). In addition, the extranuclear,cold-sensitive mutant (cs67) has been isolated,which grows at a normal rate at 37 C butmarkedly slower than the wild-type strain at20 C (23, 30), and the stable recombinant(oliAl,cs67) has been obtained, which growsslower than either of the mutants at 20 C (23).Whereas petite mutants of S. cerevisiae are

unable to grow on nonfermentable substrates,having lost all respiratory ability (6), the mater-nally inherited mi-i mutant of N. crassa is ableto respire and grows slowly exhibiting an abnor-mal cytochrome spectrum (8, 15, 20, 28). As inhigher plants (24) N. crassa possesses twoterminal respiratory pathways, one sensitive tocyanide involving the cytochromes and onesensitive to hydroxamates, and this alternative,cyanide-resistant pathway forms a substantial

proportion of total cell respiration in the mi-imutant (14, 16, 26, 28).

Cyanide-resistant respiration and its stimula-tion by growth of the organism in the presenceof antimycin A has also been reported in Asper-gillus oryzae (10, 11).

This paper describes cytochrome alterationsand cyanide-resistant respiration in the twoextranuclear mutants ofA. nidulans (oliAl) and(cs67) and in the recombinant formed fromthem and is the first report of extrachromoso-mally inherited cold sensitivity affecting mito-chondrial function. The results strengthen thehypothesis that these extranuclear mutationsare located in the mitochondrial deoxyribonu-cleic acid.

MATERIALS AND METHODSStrains and cultures. The following strains were

used and are fully described elsewhere (23): R21pabaAl,yA2 (+,+), R153 wA3, pyroA4 (+,+), OR6pabaAl, yA2 (oliAl,+), B3 wA3;pyroA4 (oliAl,+),CS67-6 wA3; pyroA4 (+,cs67), B101 wA3; pyroA4(oliAI, cs67).

Cytoplasmic genes are denoted in parentheses: oli,resistant to oligomycin; cs, cold sensitive. p-Aminobenzoic acid-requiring strains were not used inexperiments involving growth at 20 C owing to aninability to satisfy fully their requirement at thistemperature.

Maintenance of the strains, conidial production,and media are described elsewhere (21, 23). For

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390 TURNER AND ROWLANDS

growth curves, measurement of respiration, and pro-duction of mycelium for isolation of mitochondria, theorganism was cultured in 5 liters of minimal medium,containing the required supplements, in 10-liter flaskswith forced aeration (8 liters/min) at either 37 or 20 C.Cultures were inoculated with 106 conidia/ml.Measurement of respiration. Respiration of whole

mycelium was measured polarographically in a 5-mlreaction vessel at either 30 C (for 37 C-grown cul-tures) or 20 C (for 20 C-grown cultures). Inhibitorswere added to the vessel as the following solutions:NaCN, 0.1 M solution neutralized with HCl; salicylhydroxamic acid, 0.1 M either in ethanol or inaqueous solution as the sodium salt; antimycin A inethanol, 1 mg/ml. Mycelium from very young cul-tures was concentrated by centrifugation for 2 min at1,500 x g in a bench centrifuge and suspended ingrowth medium.Dry weight measurements. Samples of culture

(100 ml) were siphoned from the flasks and filteredthrough either 2.1-cm-diameter Whatman GF/Cglass-fiber disks (young cultures) or 5.5-cm-diameterWhatman no. 1 filter papers (older cultures). Themycelium was washed with 100 ml of distilled waterand dried overnight at 105 C before weighing.

Isolation of mitochondria. Approximately 100 g(wet weight) of mycelium from late exponential-phasecultures was collected by filtration through one layerof fine muslin and washed with cold tapwater, colddistilled water, and cold isolation buffer (10 mMtris(hydroxymethyl)aminomethane - hydrochloride, 1mM ethylenediaminetetraacetate, 0.5 M mannitol,pH 7.0). After each wash, the mycelium was squeezedto remove excess liquid using a Dispos-a-glove (Ethi-con Ltd., Edinburgh, U.K.). Breakage of the myceliumand isolation of mitochondria were carried out at 4 C.The mycelium was resuspended in 10 volumes ofisolation buffer and disrupted in a grinding mill (31).The effluent from the mill was filtered through eightlayers of fine muslin, and the muslin was squeezed tomaximize the yield of homogenate. The homogenatewas centrifuged at 11,000 x gav for 30 min to concen-trate the subcellular particles, which were resus-pended in 100 ml of isolation buffer and centrifuged at500 x g for 10 min to remove mycelial fragments andconidia. The supernatant was carefully removed fromthe pellet and recentrifuged at 12,000 x gav for 30 minto obtain a crude mitochondrial pellet. This was useddirectly for determination of cytochrome spectra afterresuspension in 0.5 M sucrose, 10 mM tris(hydrox-ymethyl)aminomethane - hydrochloride, 10 mMKH2PO4, 1 mM MgSO4, pH 7.0.Cytochrome spectra. Spectra (dithionite-reduced

minus oxidized) of whole mycelium were examined atroom temperature in a Unicam SP1800B spectropho-tometer after homogenization as described previously(22).

Spectra of isolated mitochondria were examined at77 K (in liquid N.) in a split-beam spectrophotometer(9) with a slit width of 0.15 mm. The cuvettes (pathlength, 2 mm) contained 0.6 ml of suspension.

Protein was determined by the method of Lowry etal. (18).

RESULTSRoom-temperature spectra of whole myce-

lium. Spectra (dithionite-reduced minus oxi-dized) of mycelium from exponential-phase cul-tures are shown in Fig. 1. Spectra of (+,cs67)grown at 37 C and (+,+) grown at 20 C were

identical to that of (+,+) grown at 37 C, andthe spectra of (oliAl,cs67) grown at 37 C and(oliAl, +) grown at 20 C were identical to that of(oliAl,+) grown at 37 C. It can be seen thatboth (+,cs67) and (oliAl,cs67) have alteredspectra in comparison to the wild-type strainwhen grown at 20 C, the most marked differ-

552560

608

1A

= 0-0j 2

3

4

A.

500 550 600 650

WAVELENGTH (nm)FIG. 1. Room-temperature difference spectra (di-

thionite-reduced minus oxidized) of mycelium. (1)(+,+): 20 h at 37 C, 4.5 mg of protein per ml; (2)(oliAl, +): 27 h at 37 C, 4.5 mg of protein per ml; (3)(+,cs67): 96 h at 20 C, 4.1 mg of protein per ml; (4)(oliAl,cs67): 120 h at 20 C, 4.2 mg of protein per ml.

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EXTRANUCLEAR MUTANTS OF A. NIDULANS

ence being the lowered concentration of cyto-chrome a,a3 (608 nm) in (+,cs67) and theincrease in concentration of cytochrome c (552nm) in both strains. This increase is greater inthe recombinant (oliAl,cs67) than in the singlemutant (+,cs67). In (+,cs67) there is also anapparent loss of cytochrome b (shoulder at 560nm), although it is visible again in the doublemutant.Although there is an alteration to the cyto-

chrome spectrum in (oliAl, +) as previouslyreported (22), this is not so marked as in thecold-sensitive mutants, and it was observedthat the abnormality was not so obvious in oldermycelium. Consequently, the spectra of(oliAl,+) and (+,+) were examined from cul-tures of different ages grown at 37 C (Fig. 2). Itcan be seen that in no case was there any loss ofcytochrome relative to the wild-type strain, butrather there was an increase in the amount ofcytochrome c in the mutant mycelium. It canalso be seen that the cytochrome concentrationsand proportions varied throughout the growthcycle in both strains, the excess of cytochrome cin ( oliAl,+) being more marked in youngercultures. Despite this, it is clear that the mu-tant contains unusually high amounts of cyto-chrome c throughout the growth cycle.Liquid N2 spectra of isolated mitochondria.

Although the room-temperature spectra showcytochrome alterations in the mutant strains,cytochrome b was not clearly resolved. Figures 3through 5 show low-temperature spectra carriedout on isolated mitochondria, in which thevarious components are more clearly resolved.The dithionite-reduced minus oxidized spec-

tra are shown in Fig. 3. In the wild-type strain,cytochrome b has been resolved into three

c

JV

555

55 5595 606

A A= 0-05

2 /

3 )

4/500 550 6901

650WAVELENGTH (nm)

FIG. 3. Low-temperature spectra (dithionite-reduced minus oxidized) of isolated mitochondria. (1)(+,+) grown at 37 C, 4.7 mg of protein per ml; (2)(oliAI,+) grown at 37 C, 5.1 mg of protein per ml; (3)(+,cs67) grown at 20 C, 5.6 mg of protein per ml; (4)(oliAl,cs67) grown at 20 C, 4.0 mg of protein per ml.

0,6-c

10-3

9O02

0.1to-1

8 O

8o

b Ab

aa3

10 20TIME (h)

FIG. 2. Cytochrome conte(oliAl,+) mycelium during theHomogenized mycelium was

mately 5 mg of protein per ml ircoefficients used were those of(17). Symbols: (0) (+,+); (x)

_' cra-bands at 555, 559.5, and 564.5 nm, whichcould reflect the presence of three differentcytochrome b species, and cytochrome cl isprobably responsible for the shoulder at 552 nm.The a-band of cytochrome c is visible at 521 nm,

30 140 50 the remaining two bands probably being due to

,nt of (+,+) and cytochrome(s) b. The increase in the proportiongrowth cycle at 37 C. of cytochrome c (548.5-nm a-band) is clearlyadjusted to approxi- visible in the mutant mitochondria, being mostn all cases. Extinction extreme in (oliAl,cs67). No corresponding in-Chance and Williams crease in cytochrome cl has occurred, resulting(oliAl, +). in its complete overshadowing in (oliAl,cs67) by

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392 TURNER AND ROWLANDS

cytochrome c. Cytochrome a,a8, (606 nm) ap- 555 559appears to be greatly diminished in (+,cs67)and, to a lesser extent, in the recombinant. J64 AThe nature of the shoulders at approximately 10.02575, 586, and 596 nm, most clearly visible in

606

5525548-5/ 5 1JA

519 |; I_002 500 SS0 600 0

1 S28 1 ^ tS§S 60 WAVELENGTH ()2

= 0.01

J\s~~~~~~~I(>0562

5293537

JAA2 1=0.02

4552

519 =0.02 500 550 600 650t 595 1-

528 ~9-5 581604 WAVELENGTH (rim)K v1 AS,575 FIG. 5. Low-temperature spectra (succinate- plus'575A/Irsantimycin A-aerated minus oxidized) of isolated mi-

tochondria. (1) (+,+): 5.1 mg of protein per ml; (2)3 (oliAI,+): 4.6 mg of protein per ml; (3) (+,cs67): 4.6

AA|,mg of protein per ml; (4) (oliAl,cs67): 3.6 mg of

JAA protein per ml. Growth conditions were as in Fig. 3.0-02 Succinate (10 mM), adenosine 5'-diphosphate (2

mM), and antimycin A (20 Ag/ml) were added to thetest sample, which was vigorously aerated beforefreezing.

4/ 5\,J L / the substrate-reduced minus oxidized spectra ofthe cold-sensitive strains (Fig. 4), is unknown.

500 550 600 650 Succinate-reduced (anaerobic) minus oxi-dized spectra are shown in Fig. 4. CytochromeWAVELENGTH (nm) b,64 was not strongly reduced by substrate, nor

FIG. 4. Low-temperature spectra (succinate- was the ,B-band at 537 nm, unless antimycin Areduced anaerobic minus oxidized) of isolated mito- was present (Fig. 5). Although no problems werechondria. (1) (+,+): 4.3 mg of protein per ml; (2) encountered with mitochondria from other(oliAl,+): 4.6 mg of protein per ml; (3) (+,cs67): 4.9 strains it was not ossible to achieve anaerobio-mg of protein per ml; (4) (oliAl,cs67): 3.4 mg of siswth mi to (olic6 ina an

protein per ml. Growth conditions were as in Fig. 3. ss with mitochondria from (oliA,cs67) in anSuccinate (10 mM) and adenosine 5'-diphosphate (2 open cuvette. Mitochondria were first allowedmM) were added to the test sample, which was to respire in the presence of 5 mM succinate andallowed to become anaerobic before freezing. 2 mM adenosine 5'-diphosphate until anaerobi-

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EXTRANUCLEAR MUTANTS OF A. NIDULANS 393

osis had been reached in an oxygen electrodevessel (1.2 ml) and were then transferred bymeans of a nitrogen-flushed syringe to a nitro-gen-flushed, air-tight cuvette (path length, 2mm). The "oxidized" suspension was vigorouslyshaken before transfer to the reference cuvette,and both suspensions were rapidly frozen inliquid N2.

Addition of antimycin A to the succinate-reduced mitochondria followed by re-aeration ofthe sample resulted in the spectra shown in Fig.5. It can be seen that the relative proportions ofthe cytochrome b components do not alter inthe mutants, although the room-temperaturespectra on the whole mycelium suggested someloss of total cytochrome b in (+,cs67) grown at20 C.Cyanide-resistant respiration of wild-type

and mutant strains. It has been reported thateven in wild-type N. crassa cyanide-resistantrespiration develops in stationary-phase cul-tures, and that the proportion of this alternativepathway in extranuclear mutants fluctuatesthroughout the growth cycle (3). Consequently,the cyanide-resistant respiration of wild-typeand mutant strains of Aspergillus were exam-inprl thrnilfflhniit thp arnwth evelp st. ?7 nnrl20C. Gshown ishown i]37 C ha(oliAl,c(+,cs67(oliAl,(oliAl,

5*0

E

E 1.0E

o-

I

w

0

0-01

FIG.(0) (+,cs67).

I-E

0w

0*10.

-'---Li--

0 20 40 60 80 100 120 140

TIME (h)

FIG. 7. Growth curves of strains at 20 C. Symbolsas in Fig. 6.

TABLE 1. Doubling times of strains growingexponentially at 37 and 20 C

Doubling time (h)Strain

37C 20C

(+,+) 1.9 8.3(oliAI, +) 2.6 10(+,cs67) 1.8 12(oliAl,cs67) 2.6 22

LLVUA51VUL, L W "Vt lOf ' two lesions has been Observed previously onrrowth curves at both temperatures are solid media (23). The growth rate of (oliAl, +) atn Fig. 6 and 7 and generation times are 2sois slightly).ighe th rat of (oliAI,) atn Table 1. It can be seen that (+,cs67) at 0 C is slightly higher than that of (+,cs67) atis a similar growth rate to (+, +) and thisgtemperature.'s67) to (oliAl,+). At 20 C, however, Figures 8, 9, and 10 show total and cyanide-)grows more slowly than (+,+) and resistant respiration of strains at 37 and 20 C. Incs67) grows more slowly than either the case of (+, +) (Fig. 8 and 9), it can bet) or (+,cs67). This additive effect of the seen that the respiration was greater than 99%

sensitive to cyanide during the exponentialphase of growth, cyanide-resistant respiration

0 E beginning to appear during the late exponential-,, / XX phase/early stationary phase and forming up to

0t' ^/ / 38% of the total respiration after 40 h at 37 C|- g t/ ~~~~~~and16%Y after 100 h at 20 C. (+,cs67) could not

be distinguished from (+,+) at 37 C. (oliAl,+),however, exhibited a substantial amount of

//X cyanide-resistant respiration (20 to 80%)Tgu/ throughout the growth cycle at 37 C (Fig. 8). A

x/X similar pattern was observed at 20 C (Fig. 9),although 100% cyanide-resistance was foundconsistently in very young cultures at this

X temperature only. (oliA,cs67) at 37 C was indis-tinguishable from (oliAl,+) at this tempera-ture.

l| s | |~~~~~~In contrast, the cold-sensitive strains showed0 10 20 30 40 a marked difference in respiratory behavior

when grown at 20 C (Fig. 10). Both (+,cs67) andTIME (h) (oliAl,cs67) exhibited cyanide-resistant respi-

6. Growth curves of strains at 37 C. Symbols: ration approaching 100% throughout most of the+); (x) (oliAl,+); (0) (+,cs67); (A) (oliAl,- growth cycle. Only in the stationary phase was

any sensitivity evident, and this never exceeded

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394 TURNER AND ROWLANDS

10 20TIME (h)

FIG. 8. Cyanide-resistant respir(oliA1,+) at 37 C. (Solid line)(dashed line) respiration in the icyanide. Other symbols as in Fig.

,/~~x\---

Xsss--X

---I---------a ---

60 80TIME (h)

increase in cyanide-resistant respiration if mea-surement was carried out at 20 C].

Effect of salicyl hydroxamate on cyanide-resistant respiration. In higher plants (24) andin Neurospora (14, 16), salicyl hydroxamate hasbeen used as an effective inhibitor of cyanide-

________ resistant respiration at a concentration of 1mM. The effect of this inhibitor with andwithout cyanide was tested on exponential-phase mycelium of (+,cs67) grown at 20 C, i.e.,when respiration was 100% cyanide resistant.

30 l,0 The results are presented in Table 2. Salicylhydroxamate alone, added either in ethanol or

*ation of (+,+) and as the sodium salt, pH 7.7, did not significantlyTotal respiration; inhibit respiration at 1 mM. However, it ispresence of 1 mM interesting to note that the addition of cyanide6. and salicyl hydroxamate together to the respir-

ing mycelium resulted in up to 90% inhibition ofrespiration.

Effect of antimycin A. Antimycin A addedto the mycelium at a concentration of 10 gg/ml

x (2 Ag/mg [dry weight] of mycelium) had a/ \ parallel effect to cyanide in all the strains

tested, effectively inhibiting the cyanide-sensi-tive respiration but not the alternative respira-tion.

0 X Growth yield experiments. In view of theinsensitivity of the respiration of 20 C-grown

"x _.X-- (+,cs67) to salicyl hydroxamate except in thepresence of cyanide or antimycin A, it seemedpossible that the mutants were not actually

100 120 using an alternative pathway during growth,but only under experimental conditions when

-ation of (±+) and cyanide or antimycin blocked the normal termi-Fig. 6 and 8. nal respiration. Since 100% respiration via the

alternative pathway would imply the bypassingof coupling sites II and III (16, 28), (+,cs67)

20

-7

B15

Zo,!~-Ej 0

wLO 5

5X

0 40 80TIME (h)

120 160 200

FIG. 10. Cyanide-resistant respiration of (+,cs67)and (oliA1,cs67) at 20 C. Symbols as in Fig. 6 and 8.

25%. In younger cultures, cyanide at 1 mMactually stimulated respiration by up to 11% inthe case of (+,cs67) and 19% with (oliAl,cs67).

It should be noted that the temperature usedfor the measurement of respiration did notaffect the proportion of cyanide resistance [e.g.,(+,cs67) grown at 37 C did not show any

TABLE 2. Effect of salicyl hydroxamate and cyanideon cyanide-resistant respiration of (+,cs67)

grown at 20 Ca

Respiration rate InhibitionAdditions in sequence

(nmol of of originalAdditions in sequence Oji/g rt0,/min/mg ratedry weight) (% )

1. None 19.62. Salicyl hydroxamate, 18.8 4

1 mM3. + Cvanide, 1 mM 2.2 89

1. None 19.62. Cyanide, 1 mM 19.2 23. + Salicvl hN droxa- 1.5 92

mate, 1 mM

a The mycelium (0.7 mg [dry weight Vml) was taken.from an 88-h, 20 C-grown culture. A 5-ml amount ofculture was used in each determination. Both inhibi-tors were added as aqueous solutions.

25

20

~-0

0-

015cn

, >1 o _

t7 E

W-oE5 -

-S

O _40

FIG. 9. Cyanide-resistant respir(oliA1,+) at 20 C. Symbols as in I

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EXTRANUCLEAR MUTANTS OF A. NIDULANS 395

grown at 20 C would be expected to show asubstantially lower growth yield than (+,+)under identical conditions. A growth yield de-termination on glucose was carried out as de-scribed previously (22), and the results areshown in Fig. 11. There appeared to be little orno difference in growth efficiency, suggestingthat the alternative pathway is not used by(+,cs67) in vivo to any great extent. It hasalready been reported that there is little differ-ence in growth efficiency between (oliAl, +) and(+,+) (22).

DISCUSSION

The cytochrome spectrum of wild-type A.nidulans closely resembles that reported for A.oryzae (29). Cytochrome b has been resolvedinto three a-bands analogous to those reportedin mammalian mitochondria (4, 32), higherplants (17), and in N. crassa (28), although onlytwo peaks were resolved in substrate- or dithio-nite-reduced minus oxidized spectra of thelatter organism. Enhancement of the 564-nmpeak was observed in the presence of antimycinA, coinciding with a similar effect on the bandat 537 nm, presumably the d-band of thiscomponent.The increase in the amount of cytochrome c is

noticeable in all mutant strains and has beenobserved in respiratory-deficient nuclear (5)and extranuclear (8, 15, 28) mutants of Neuro-spora, as well as in A. oryzae (29), Pythiumultimum (19), N. crassa (27), and HeLa cells(12) grown in the presence of chloramphenicol.In all these cases, it seems that some mitochon-drial regulation of cytochrome c synthesis hasbeen disrupted. Although there is some loss oftotal cytochrome b in (+,cs67) grown at 20 C,

12

E 0

Em 0-8_ ~~~~~~~~~~8

3: /

0 4

D

w

I

0 1 2GLUCOSE (mg/ml)

FIG. 11. Growth yields of (+, +) and (+,cs67) on

glucose at 20 C. Symbols: (a) (+,+); (0) (+,cs67).

the antimycin A spectra on isolated mitochon-dria suggest that there is no alteration in theratio of the cytochrome b components in any ofthe mutants. von Jagow et al. (28) claimed thatthe ratio of cytochrome b components variedfrom the wild-type ratio in the mi-i mutant ofN. crassa, although this has been disputed byLambowitz and Bonner (13).A high level of cyanide-resistant respiration

appears to be a regular feature of respiratory-deficient mutants in N. crassa, whether themutation is nuclear or extranuclear (5, 14), andis also apparent when mitochondrial function isdisrupted by growth of the organism with anelectron transport inhibitor (14) or a mitochon-drial protein synthesis inhibitor (14, 16, 27).Antimycin A- or cyanide-resistant respirationhas also been observed in A. oryzae (10) andEuglena gracilis (25) grown in the presence ofantimycin. It has been suggested that the alter-native pathway synthesized under such condi-tions permits reoxidation of reduced nucleotidesand allows continuation of glycolysis and sub-strate-level phosphorylation when the normalterminal cytochrome pathway is defective (16).This explanation cannot fully apply to A. nidu-lans in view of the inhibitor studies and growthyield experiments, which imply that the alter-native pathway, although present in (+,cs67)grown at 20 C, may not be operative. In addi-tion, the fact that all the cytochromes arereduced by succinate in the mutant strains, andthat antimycin A prevents reoxidation of the bcytochromes while allowing reoxidation of cyto-chromes cl, c, and a,a,, support the view thatthe cytochrome pathway is functional in allmutant strains and that this pathway is an-timycin sensitive as in the wild type. It isinteresting to note that similar results for re-sponse to the inhibitors salicyl hydroxamateand cyanide have been observed by Edwardsand Kwiecinski (5) in the nuclear respiratory-deficient mutant cni-1 of Neurospora, contrast-ing with the behavior of mi-i, in which salicylhydroxamate inhibits the alternative pathwayin the absence of cyanide. The authors con-cluded that either pathway was capable oftaking the full electron flux in the cni-1 mutant,whereas only the cyanide-resistant pathway wasable to do this in mi-1. The nature of thealternative oxidase is still not known, but it hasbeen suggested that the branch point from thenormal pathway lies in the region of ubiqui-none, bypassing sites II and III (16, 28). Studieson isolated mitochondria of A. nidulans are inprogress to investigate further the behavior androle of this alternative pathway in the extranu-clear mutants.

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396 TURNER AND ROWLANDS

To date, no differences have been observedbetween (+,cs67) grown at 37 C and the wild-type strains. Cold sensitivity of this mutanttogether with the cytochrome alterations at20 C suggested that this organism might havesome mitochrondrial ribosome assembly defect,although labeling experiments (unpublisheddata) have failed to show any such defect andthe nature of the cold sensitivity is not yetunderstood.The oligomycin-resistant properties of

(oliAl,+) have already been described (22), butthe mutation conferring oligomycin resistanceseems also to cause some defect in electrontransport, resulting in excess production ofcytochrome c, stimulation of cyanide-resistantrespiration, and slow growth of the organism. Aspreviously stated (22), after many crosses, theslow-growth character has never been separatedfrom oligomycin resistance, and both may bedue to the alteration of a single, mitochondriallycoded protein component of the inner mem-

brane.

ACKNOWLEDGMENTS

We wish to thank C. Watson for her technical assistanceand O.T.G. Jones for use of the split-beam spectrophotome-ter.

This investigation was supported by a Science ResearchCouncil grant (G.T.) and a Beit Memorial Research Fellow-ship (R.T.R).

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8. Haskins, F. A., A. Tissieres, H. K. Mitchell, and M. B.Mitchell. 1953. Cytochromes and the succinic acidoxidase system of poky strains of Neurospora.

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