Mechanisms of Ageing and Development, 55 (1990) 15--37
15
Elsevier Scientific Publishers Ireland Ltd.
B I O C H E M I C A L , G E N E T I C A N D U L T R A S T R U C T U R A L D E F E C T S IN A M I T O C H O N D R I A L M U T A N T (ER-3) OF N E U R O S P O R A C R A S S A W I T H SENESCENCE P H E N O T Y P E
F R A N K D. N I A G R O * and N.C. M I S H R A Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208 (U.S.A.)
(ReceivedNovember 6th, 1989) (Revision receivedJanuary 23rd, 1990)
SUMMARY The structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant E R - 3 of Neurospora crassa are described. The mitochondrial mutant, which grows at a rate of only 10070 of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa 3. Cytochrome oxidase activity in the mutant mitochondria was only about 5°70 of the wild type mitochondria. However, the total whole cell respiration rate of the mutant was 33070 greater than that of the wild type, while the cyanide-resistant respiration rates were equal. The results of inhibitor studies clearly demonstrate that the mutant possesses a defect in one or more components of the terminal oxidase. Electron microscopic examination of whole cell sections and subsequent morphometric analysis revealed a significant (33070) reduction in membrane surface density of mitochondrial cristae in the mutant as compared with the wild type. Results of genetic and heterokaryon analyses indicate the location of mutation (ER-3) in the mitochondrial DNA. It is concluded that the senescence mutant E R - 3 possesses a defect in the terminal portion of the mit0chondrial respiratory apparatus. These results are consistent with previous analyses of mitochondrial D N A populations, and support the notion that obligately aerobic eukaryotic cells deficient in mitochondrial respiration necessarily exist as a result of stable heteroplasmosis and that defects in mitochondria lead to senescence in N e u r o s p o r a mutant E R - 3 . K e y words: Induced sensescence; Mutation; Mitochondria; Cytochromes; Ultras-
tructure Address all correspondence to: N.C. Mishra. *Present address: Department of Medical Microbiology, College of Veterinary Medicine, University of
Georgia, Athens, GA 30602, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990Elsevier Scientific Publishers Ireland Ltd.
16
INTRODUCTION
Mitochondrial structure and function are controlled by the combined expression of nuclear and mitochondrial genes [1]. These gene products include one or more components of the mitochondrial electron transport chain. Mutations which produce defects in the mitochondrial respiratory chain, if not lethal, normally yield strains with abnormal pleiotropic phenotypes. Studies of several mitochondrial mutants have been useful in determining the role of mitochondrial gene products in development of an organism. One such group of mutants of Neurospora, called stopper, are characterized by the absence of cytochromes and cytochrome oxidase concomitant with the deletion of the mitochondrial chromosome [2--5]. Characterization of the Neurospora mitochondrial electron transport system has revealed the presence of one or more alternate oxidases, which allow this obligately aerobic fungus to grow even in the presence of terminal oxidase inhibitors [6] or, presumably, defects in the cyanide-sensitive terminal oxidase [7]. Mixed populations of mitochondria, with different susceptibilities to an inhibitor of the alternate oxidase, have been purified f r o m the Neurospora poky mutant [8]. This heteroplasmosis may represent the normal state of affairs for mycelia containing mitochondria with defective components. In fact, mixed populations of mitochondrial DNA have been demonstrated in a stopper mutant [10,11]. Evidence has been presented that mitochondrial D N A from such a stopper mutant lacks two protein genes required for proper assembly of mitochondrial complexes I, III and IV [9]. We have recently described a new stopper mutant (ER-3) of Neurospora crassa induced by ethidium bromide [10,11]. This mutant displays the intermittent growth phenotype characteristic of the other mitochondrial stopper mutants [2], and, in addition, a senescence phenomenon similar to the kalilo strains of Neurospora intermedia and those of other filamentous fungi [12,13]. Analysis of the ER-3 mitochondrial chromosome reveals that this senescence mutant contains a mixed population of mitochondrial DNAs. The defective population of mitochondrial D N A molecules contains a deletion of the mitochondrial genes coding for at least two components of the mitochondrial respiratory system [11]. In an effort to better understand the role of mitochondrial genes in determining mitochondrial structure and function, we have extended the previous analysis of the ER-3 strain to correlate the deletion of mitochondrial genes with loss of mitochondrial respiratory function. In addition, mitochondrial ultrastructure was examined in order to determine whether quantifiable structural changes were associated with altered biochemical function at the organelle level. The results of these studies with ER-3 represent the first demonstration of associated biochemical and ultrastructural lesions in a known mitochondrial mutant. These data also support the idea that defects in mitochondria may result in a form of cellular senescence. MATERIALS AND METHODS
Strain, media and linear growth measurement The wild type RL3-8A was grown on Vogel's minimal medium with 2°7o sucrose.
17 The mutant ER-3 was grown on potato dextrose medium (Difco), as described previously, either in liquid shake cultures (100--125 rev./min) or on solid media (2°70 agar). Linear growth was measured in growth tubes approximately 600 mm long by the method of Ryan et ai. [14].
Genetic analysis Reciprocal crosses between the mutant ER-3 with nuclear ini (inositol) marker and normal strain with trp-3 (tryptophan) marker were attempted on synthetic crossing medium. Progeny were scored for nutritional auxotrophy (nuclear markers) and growth phenotype (normal or slow) in growth tubes containing potato dextrose agar. Heterokaryons were forced on Vogel's minimal agar slants with a normal strain carrying the pan-2 (pantothenic acid) marker. Individual conidial isolates were scored for their growth phenotype (normal or slow). Preparation o f mitochondria Mitochondria were isolated by a modification of flotation methods as used in our laboratory [10,11]. Cytochrome spectra Room temperature cytochrome difference spectra were performed according to published procedures [ 15]. Whole cell respiration studies Whole cell respiration rates were determined using a Clark type oxygen electrode (Yellow Springs Instruments) to monitor oxygen uptake by actively growing cells in liquid growth medium. Liquid shake cultures (50 ml) were grown in potato dextrose medium at 30°C for 24--36 h. Mycelial masses were removed from the culture flasks, rinsed quickly in sterile distilled water (at 30°C), and placed into a reaction vessel. The reaction vessel contained 5 ml of fresh potato dextrose medium. For determination of cyanide insensitive respiration rates, 5/al of freshly prepared 1 M KCN was added to the reaction vessel immediately prior to addition of mycelia. Oxygen uptake was monitored at 30°C by recording 070 oxygen readings at 5-s intervals for 1 min. Calculations of respiratory activity in atoms of oxygen/l per mg dry weight were performed according to published procedures using a value of 238 /amol/l for air saturated water [16]. Other biochemical assays Cytochrome oxidase activity was determined by a modification of published methods [17]. Protein was determined using the procedure of Lowry et al. [18]. Electron microscopy o f mycelium and morphometric analysis Preparation of cells for electron microscopy was by a modification of published procedures [19]. Samples of mycelia were removed from mid-log phase (36--48 h)
18 cultures; fixed sections were stained with uranyl acetate and lead citrate and examined at 80 kV. Only sections showing no knife marks, compression or other distortion were used for morphometry. All micrographs were made at one sitting to minimize any deviation in magnification due to voltage fluctuations, etc. A double lattice was printed on each image by overlaying a grid on the photographic paper during the exposure. For volumetric analysis the intersections of lines in the double-lattice were used as test points. Test points were scored as falling within the images of nuclei, mitochondria or cytoplasm on the micrographs. Fractional volumes were derived as percentages of the total number of test points counted. Membrane surface densities were obtained by counting the intersections of one lattice of test lines with the images of nuclear envelopes or mitochondrial inner and outer membranes. The second lattice served to divide the test line lattice into unit lengths for deriving comparative data. Membrane surface densities were calculated using the following formula: membrane surface density = 2NL~ where NL~ is the number of membrane intersections per unit length of sampling line [20]. Standard deviations were calculated as the deviation among the values calculated for each of the sections examined.
Purification o f mitochondrial DNA (mtDNA) and visualization by electron microscopy The m t D N A was prepared by methods described previously as practiced in our laboratory [10,11] and prepared for electron microscopy according to established procedures [21]. Stained grids were rotary shadowed with uranium oxide and viewed with a Jeol 100-B electron microscope. Lengths of D N A molecules were calculated from measurements of photographic enlargements made with a map measurer. RESULTS
Growth characteristics and senescence phenotype o f the mutant (ER-3) and its heterokaryon, and genetic analysis in sexual crosses The ethidium bromide induced mutant, ER-3, was first identified by its small colony size on sorbose containing medium. Later the growth characteristics of ER-3 were examined in race tubes and the mutant was found to grow much more slowly than the wild type (Fig. 1A). The growth pattern of ER-3 was found to be similar to that of the other previously described Neurospora stopper mutants in that the periods of slow mycelial growth were interrupted by periods of no growth (Fig. 1A). However, the growth pattern of ER-3 differed from the previously described stopper mutants in that ER-3 ceased to grow after one to several rounds of interrupted growth (Fig. IB). In many cases stopped periods were quite extensive, often exceeding 100 h. The stopping phenomenon in ER-3 was nearly the same as described for Abnormal-I and other stopper mutants in that the resumption of growth did not occur from the hyphal tips, but rather from an area several millimeters back from
DISTANCE
(ram)
0
- E ~ ~,~
I I
I I
.~. =. ~.~ ~ ~'~.~
i i i i i i i
~" ~ ~,~ ~• ~ ; ~
=" .~. ="
ilOwl~
m
IUUl
t
!
ill
~.
!
I
O"
IIiit
E.. DISTANCE
~ . ~
o
o . ~ =o"
~B ~o ='~..~ =-. a
.-,I
+5,~
i o~.~ 61
~
~
(ram)
~
~
§
20 the growth front. However, in m a n y experiments, E R - 3 displayed an apparent senescence, failing to resume growth even after 300 h (Fig. 1B). In E R - 3 , the senescence phenotype was often accompanied by the formation of droplets of a brown liquid on the mycelia near the growth frontier. Immediately following senescence, the medium distal to the growth front was found to darken, presumably due to the discharge of this brown liquid into the growth medium. This phenomenon was also seen when E R - 3 became senescent in liquid culture with a progressive darkening of the liquid medium shortly following cessation of growth. A heterokaryon was forced between E R - 3 [carrying a mutation for inositol auxotrophy (inl-)], and strain 2248 (a p a n - 2 mutant with normal growth and cytochrome content). Neither E R - 3 nor strain 2248 grew on minimal medium; however, the heterokaryon grew slowly at a rate significantly less than that of the wild type (Fig. 1A). The growth characteristics of the heterokaryon were much different than that of E R - 3 . In the heterokaryon, the stop-start pattern of linear growth was not observed in 500 h of growth. In addition, none o f the heterokaryons formed exhibited the senescence phenomenon. Also unlike the E R - 3 mutant, the heterokaryons produced conidia. A representative analysis o f conidia f r o m one heterokaryon presented in Table I and Fig. 1C showed two discrete groups: fast (wild type growth rate (approx. 3.8 m m / h ) , or slow (0.56 m m / h ) growers). However, the slow growing condial isolates grew at rates faster than the original E R - 3 mutant. Thus, the fast and slow growing isolates m a y represent the segregation of the normal and respiratory deficient mitochondria during the breakdown o f heteroplasmy. Also, the heterokaryon showed a wild type cytochrome spectrum. These data show that the heterokaryon behaved like the wild type strain. Thus, the suppressiveness in E R - 3 is apparently different from that described in other stopper mutants [2]. With the mutant determinant in the heterokaryon, it was of further interest to see if the heterokaryon could act as a protoperithecial parent in sexual crosses. If so, it might be possible to directly demonstrate maternal transmission to the progeny. Table II shows the result of such reciprocal crosses. When the heterokaryon acted as
TABLE I SEGREGATION OF NORMAL AND SLOW GROWING PHENOTYPES AMONG CONIDIAL ISOLATES FROM THE HETEROKARYONOF THE WILD TYPE AND STOPPER STRAINS Heterokaryon:
ER-3 (0.4) ~
Pan-2 (3.8) u
A In I- Pan [ - ]
A Inl*Pan-[+]
Growth phenotypes o f Pan-2 conidial isolates Normal Slow
Total
11 ( 3 . 8 ) a
75
64 (0.56) a
aGrowth rate (mm/h) indicated in parentheses.
21
TABLE II SEGREGATION OF NORMAL AND SLOW GROWING PHENOTYPES AMONG PROGENY OF THE CROSS BETWEEN WILD TYPE AND ER-3 (FIRST CROSS IN THIS TABLE) OR BETWEEN HETEROKARYONS CONTAINING THE STOPPER DETERMINANT (ER-3) AND THE WILD TYPE STRAIN (SECOND AND THIRD CROSSES IN THIS TABLE) Cross
Total
Ascospores
ascospores
germinated
Normal growers
SIow growers
240
94 (39.1 °70)
94
0
269
90 (33.5070)
85
5
224
24 (10.7070)
24
0
isolated 1. a Inl* Trp- [ + ]
x lnl- Trp ÷ [ - ] 2. a Inl* Pan* Trp- [ + ]
x A A 3. A A
InI- P a n ÷ Trp ÷ [ lnl* P a n - Trp ÷ [ + lnl" Pan" Trp ÷ [ I n l +P a n - Trp ÷ [ +
] + ] ] +
]
× a Inl +P a n ÷ Trp- [ + ]
The first member of each cross is the protoperithecial parent. Percent germination in parentheses.
the conidial parent (cross #2), the results were essentially identical to the cross involving ER-3 alone (cross * 1). In this experiment the tryptophan requiring mutant trp-3 (with wild type growth) was used as the perithecial parent and, as expected, almost all (94%) of the progeny were normal (wild type) growers. If, however, the heterokaryon was used as the perithecial parent, the progeny were again all wild type growers and none were slow growers. In all crosses performed with ER-3, the ascospore germination rate was exceedingly low. The ascospore germination was 33.5°70 when the heterokaryon was the conidial parent (cross #2) but only 10.7070 when it was the perithecial parent (cross #3). Thus, the result of sexual crosses involving ER-3 were similar to those reported for certain other stopper mutants [22].
Mitochondrial cytochrome content The senescence mutant ER-3 was found to possess abnormal levels of mitochondrial cytochromes (Fig. 2). The room temperature cytochrome spectrum of the mutant shows that the alpha cyt a + a 3 absorption peak at 610 nm is completely absent. However, this is in sharp contrast to the picture portrayed by the cytochrome soret bands. While the absorption maximum in the soret region is the same for both the wild type and ER-3, at 430 nm (not shown), the ratios of the gamma peak at 443 nm (cyt a 3) to that at 430 nm (cyt b) indicate that some component of cytochrome a is still present (Table III).
22
0.011
ER-3A
O Z 4( W
HK
Port °2A
500
550
600
650
WAVELENGTH (am)
Fig. 2. Oxidized versus reduced cytochrome difference spectra of the wild type (pan-2A), the senescence mutant (ER-3) and heterokaryon between them. Mitochondrial fractions were prepared, sonicated and solubilized with sodium deoxycholate. Sodium dithionite was added and the reduced spectra measured from 480 to 650 nm. Protein concentrations of preparations are: p a n - 2 A , 5.6 mg/ml; E R - 3 , 6.6 rag/ ml; heterokaryon 4.4 mg/ml.
TABLE II1 RELATIVE SPECIFIC CYTOCHROME MUTANT M I T O C H O N D R I A a Strain
CONTENTS OF WILD TYPE AND SENESCENCE
Cytochrome content ~
Soret
A 430 cyt b
cyt c
cyt a + a3
A46o RL3-SA ER-3A
0.0064 (0.0028) 0.0030 (0.0003)
0.0067 (0.0051) 0.0052 (0.0010)
0.0014 (0.0004) 0
0.015 0.018
=Cytochrome contents expressed as absorbance/mg per ml of mitochondrial protein. Values in parentheses are standard errors. Values for cytochromes b, c and a + a~ were derived as described in Methods from cytochrome alpha peaks. The soret band ratio for cyt a + a~ is derived from gamma peak values at 443 nm compared to absorbances at 460 nm.
23 The amounts of mitochondrial cytochromes from E R - 3 and the wild type strain RL3-8A presented in Table III indicate that the specific amounts of all three mitochondrial cytochromes are reduced in the mutant. In addition, the proportion of cytochrome b to cytochrome c is reduced by almost 40% as compared with that of the wild type. These data are consistent with the previously described stopper and poky mutants of N . crassa [6,22]. C y t o c h r o m e oxidase a n d respiration deficiency
In order to determine the functional relationship between the observed cytochrome deficiencies and the severely impaired growth rate of the mutant (0.398 _+ 0.032 m m / h ) versus the wild type (3.78 ___ 0.16 mm/h), two types of experiments were conducted. For the first, mitochondria were purified on sucrose gradients and cytochrome oxidase activity determined by means of a spectrophotometric assay. The results (Table IV) indicate that the specific activity of this enzyme in the mutant is only approximately 5% of that in the wild type. Table IV also portrays the results of the second series of experiments in which whole cell respiration rates were used as a measure of mitochondrial impairment. Whole cells were placed in fresh liquid growth medium in a Clark oxygen electrode and oxygen uptake was measured both with and without potassium cyanide present in the medium. The cyanide sensitive respiration rates appear to be identical for both the mutant and wild type, at approximately 12.5/amol of oxygen consumed/h per mg dry weight of mycelia. However, the total cell respiration rate of E R - 3 is 1.3 times greater than that of the wild type. Thus, the cyanide-resistant respiration represents a smaller fraction of total respiration in the mutant (17.5%) than in the wild type (22.5%).
TABLE IV CYTOCHROME OXIDASE AND WHOLE CELL RESPIRATION RATES OF THE WILD TYPE AND SENESCENCEMUTANT' Strain
RL3-8A ER-3A
Cytochrome c oxidase
61.8 (5.3) 3.1 (0.4)
Whole cell respiration Total
KCN resistant
54.41 (9.06) 72.66 (7.45)
12.42 (3.43) 12.72 (2.99)
•Cytochrome c oxidase values are expressed as a change in absorbance/min per milligram of mitochondrial protein and are the averageof two trials. Whilecell respiration rates are expressed as micromolesof oxygen consumed/h per mg of dry weight of cells. Values for RL3-8A are the averages from three trials and those for ER-3A are averagesof four trials. Valuesin parentheses are standard errors.
24
Effect o f respiratory inhibitors Stopper mutants are characterized by the unique stop-start growth phenotype displayed by mycelia grown on solid media. When such stopper mutants are grown in race tubes [14] and their linear growth measured over the course of time, the stopstart pattern is readily apparent (Fig. 2). The processes responsible for this abnormal growth pattern are not fully understood, however, it was of considerable interest to use this trait as a measure of respiratory sufficiency in growing cells. It was hoped that it would be possible to provide evidence for a biochemical defect in vivo by growing the mutant in the presence of respiratory inhibitors and examining the effect on the mutant growth pattern. The concentrations of inhibitors used in these studies were taken f r o m previously published studies of Neurospora grown in liquid culture [19--20]. It was also desired that growth of the wild type strain in the presence of such inhibitors would induce respiratory deficient or stopper phenocopies. The generation of such phenocopies could provide additional information concerning the identification of the stopper biochemical defect in vivo. The results of these experiments are shown in Fig. 3. The typical growth pattern for wild type is that of the control (Fig. 3A). When the wild type was grown in the presence of 1 m M cyanide, its initial lag phase was somewhat lengthened as compared to that in the absence of inhibitor. However, after 2 days a normal growth rate was resumed (Fig. 3A). Growth in the presence of salicyl hydroxamic acid (SHAM), an inhibitor of the Neurospora cyanide-resistant alternate oxidase, was initially comparable to that in the absence of inhibitor, but progressively slowed until it attained a rate approximately one fourth that of the control. Growth in the presence of cyanide and S H A M together produced a pattern which mirrored that obtained in the presence of S H A M alone. However, the additional effect of cyanide was again evident as a lag during the initial 24 h of growth (Fig. 3A). The effect of sodium azide on the growth of the wild type was even more pronounced. Wild type mycelia grown in the presence of azide together with either cyanide or S H A M grew at a rate comparable to that of the mutant (ER-3). However, in the presence of azide, cyanide had a much more inhibitory effect on growth than S H A M (Fig. 3A). Figure 3B shows the results typically obtained when ER-3 is grown in the presence of the same inhibitors (i.e., cyanide, S H A M and sodium azide). As with the wild type, growth of ER-3 in the presence of 1 mM cyanide is slowed as compared with the rate of growth in the absence o f the inhibitor. ER-3 did not grow in the presence of either S H A M or sodium azide (Fig. 3B). The second set of inhibition studies involved antimycin A, oligomycin and the uncoupler 2,4-dinitrophenol (Figs. 3C and 3D). The only apparent effect of those compounds on the wild type was a somewhat longer lag in initial growth and a slightly reduced rate o f growth compared to that in the absence of inhibitors (compare Figs. 3A and 3C). However, the mutant was unable to grow beyond germination in the presence of the uncoupler (Fig. 3D).
25
50O
400
400
300
~3OO
200
¢:
2OO
I00 I00
IOO
200
I
300
I00
200 TIME
TIME (hours) SO0
5001
400
40C
300
"~300
300
(hours)
z
15
Elsevier Scientific Publishers Ireland Ltd.
B I O C H E M I C A L , G E N E T I C A N D U L T R A S T R U C T U R A L D E F E C T S IN A M I T O C H O N D R I A L M U T A N T (ER-3) OF N E U R O S P O R A C R A S S A W I T H SENESCENCE P H E N O T Y P E
F R A N K D. N I A G R O * and N.C. M I S H R A Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208 (U.S.A.)
(ReceivedNovember 6th, 1989) (Revision receivedJanuary 23rd, 1990)
SUMMARY The structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant E R - 3 of Neurospora crassa are described. The mitochondrial mutant, which grows at a rate of only 10070 of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa 3. Cytochrome oxidase activity in the mutant mitochondria was only about 5°70 of the wild type mitochondria. However, the total whole cell respiration rate of the mutant was 33070 greater than that of the wild type, while the cyanide-resistant respiration rates were equal. The results of inhibitor studies clearly demonstrate that the mutant possesses a defect in one or more components of the terminal oxidase. Electron microscopic examination of whole cell sections and subsequent morphometric analysis revealed a significant (33070) reduction in membrane surface density of mitochondrial cristae in the mutant as compared with the wild type. Results of genetic and heterokaryon analyses indicate the location of mutation (ER-3) in the mitochondrial DNA. It is concluded that the senescence mutant E R - 3 possesses a defect in the terminal portion of the mit0chondrial respiratory apparatus. These results are consistent with previous analyses of mitochondrial D N A populations, and support the notion that obligately aerobic eukaryotic cells deficient in mitochondrial respiration necessarily exist as a result of stable heteroplasmosis and that defects in mitochondria lead to senescence in N e u r o s p o r a mutant E R - 3 . K e y words: Induced sensescence; Mutation; Mitochondria; Cytochromes; Ultras-
tructure Address all correspondence to: N.C. Mishra. *Present address: Department of Medical Microbiology, College of Veterinary Medicine, University of
Georgia, Athens, GA 30602, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland
© 1990Elsevier Scientific Publishers Ireland Ltd.
16
INTRODUCTION
Mitochondrial structure and function are controlled by the combined expression of nuclear and mitochondrial genes [1]. These gene products include one or more components of the mitochondrial electron transport chain. Mutations which produce defects in the mitochondrial respiratory chain, if not lethal, normally yield strains with abnormal pleiotropic phenotypes. Studies of several mitochondrial mutants have been useful in determining the role of mitochondrial gene products in development of an organism. One such group of mutants of Neurospora, called stopper, are characterized by the absence of cytochromes and cytochrome oxidase concomitant with the deletion of the mitochondrial chromosome [2--5]. Characterization of the Neurospora mitochondrial electron transport system has revealed the presence of one or more alternate oxidases, which allow this obligately aerobic fungus to grow even in the presence of terminal oxidase inhibitors [6] or, presumably, defects in the cyanide-sensitive terminal oxidase [7]. Mixed populations of mitochondria, with different susceptibilities to an inhibitor of the alternate oxidase, have been purified f r o m the Neurospora poky mutant [8]. This heteroplasmosis may represent the normal state of affairs for mycelia containing mitochondria with defective components. In fact, mixed populations of mitochondrial DNA have been demonstrated in a stopper mutant [10,11]. Evidence has been presented that mitochondrial D N A from such a stopper mutant lacks two protein genes required for proper assembly of mitochondrial complexes I, III and IV [9]. We have recently described a new stopper mutant (ER-3) of Neurospora crassa induced by ethidium bromide [10,11]. This mutant displays the intermittent growth phenotype characteristic of the other mitochondrial stopper mutants [2], and, in addition, a senescence phenomenon similar to the kalilo strains of Neurospora intermedia and those of other filamentous fungi [12,13]. Analysis of the ER-3 mitochondrial chromosome reveals that this senescence mutant contains a mixed population of mitochondrial DNAs. The defective population of mitochondrial D N A molecules contains a deletion of the mitochondrial genes coding for at least two components of the mitochondrial respiratory system [11]. In an effort to better understand the role of mitochondrial genes in determining mitochondrial structure and function, we have extended the previous analysis of the ER-3 strain to correlate the deletion of mitochondrial genes with loss of mitochondrial respiratory function. In addition, mitochondrial ultrastructure was examined in order to determine whether quantifiable structural changes were associated with altered biochemical function at the organelle level. The results of these studies with ER-3 represent the first demonstration of associated biochemical and ultrastructural lesions in a known mitochondrial mutant. These data also support the idea that defects in mitochondria may result in a form of cellular senescence. MATERIALS AND METHODS
Strain, media and linear growth measurement The wild type RL3-8A was grown on Vogel's minimal medium with 2°7o sucrose.
17 The mutant ER-3 was grown on potato dextrose medium (Difco), as described previously, either in liquid shake cultures (100--125 rev./min) or on solid media (2°70 agar). Linear growth was measured in growth tubes approximately 600 mm long by the method of Ryan et ai. [14].
Genetic analysis Reciprocal crosses between the mutant ER-3 with nuclear ini (inositol) marker and normal strain with trp-3 (tryptophan) marker were attempted on synthetic crossing medium. Progeny were scored for nutritional auxotrophy (nuclear markers) and growth phenotype (normal or slow) in growth tubes containing potato dextrose agar. Heterokaryons were forced on Vogel's minimal agar slants with a normal strain carrying the pan-2 (pantothenic acid) marker. Individual conidial isolates were scored for their growth phenotype (normal or slow). Preparation o f mitochondria Mitochondria were isolated by a modification of flotation methods as used in our laboratory [10,11]. Cytochrome spectra Room temperature cytochrome difference spectra were performed according to published procedures [ 15]. Whole cell respiration studies Whole cell respiration rates were determined using a Clark type oxygen electrode (Yellow Springs Instruments) to monitor oxygen uptake by actively growing cells in liquid growth medium. Liquid shake cultures (50 ml) were grown in potato dextrose medium at 30°C for 24--36 h. Mycelial masses were removed from the culture flasks, rinsed quickly in sterile distilled water (at 30°C), and placed into a reaction vessel. The reaction vessel contained 5 ml of fresh potato dextrose medium. For determination of cyanide insensitive respiration rates, 5/al of freshly prepared 1 M KCN was added to the reaction vessel immediately prior to addition of mycelia. Oxygen uptake was monitored at 30°C by recording 070 oxygen readings at 5-s intervals for 1 min. Calculations of respiratory activity in atoms of oxygen/l per mg dry weight were performed according to published procedures using a value of 238 /amol/l for air saturated water [16]. Other biochemical assays Cytochrome oxidase activity was determined by a modification of published methods [17]. Protein was determined using the procedure of Lowry et al. [18]. Electron microscopy o f mycelium and morphometric analysis Preparation of cells for electron microscopy was by a modification of published procedures [19]. Samples of mycelia were removed from mid-log phase (36--48 h)
18 cultures; fixed sections were stained with uranyl acetate and lead citrate and examined at 80 kV. Only sections showing no knife marks, compression or other distortion were used for morphometry. All micrographs were made at one sitting to minimize any deviation in magnification due to voltage fluctuations, etc. A double lattice was printed on each image by overlaying a grid on the photographic paper during the exposure. For volumetric analysis the intersections of lines in the double-lattice were used as test points. Test points were scored as falling within the images of nuclei, mitochondria or cytoplasm on the micrographs. Fractional volumes were derived as percentages of the total number of test points counted. Membrane surface densities were obtained by counting the intersections of one lattice of test lines with the images of nuclear envelopes or mitochondrial inner and outer membranes. The second lattice served to divide the test line lattice into unit lengths for deriving comparative data. Membrane surface densities were calculated using the following formula: membrane surface density = 2NL~ where NL~ is the number of membrane intersections per unit length of sampling line [20]. Standard deviations were calculated as the deviation among the values calculated for each of the sections examined.
Purification o f mitochondrial DNA (mtDNA) and visualization by electron microscopy The m t D N A was prepared by methods described previously as practiced in our laboratory [10,11] and prepared for electron microscopy according to established procedures [21]. Stained grids were rotary shadowed with uranium oxide and viewed with a Jeol 100-B electron microscope. Lengths of D N A molecules were calculated from measurements of photographic enlargements made with a map measurer. RESULTS
Growth characteristics and senescence phenotype o f the mutant (ER-3) and its heterokaryon, and genetic analysis in sexual crosses The ethidium bromide induced mutant, ER-3, was first identified by its small colony size on sorbose containing medium. Later the growth characteristics of ER-3 were examined in race tubes and the mutant was found to grow much more slowly than the wild type (Fig. 1A). The growth pattern of ER-3 was found to be similar to that of the other previously described Neurospora stopper mutants in that the periods of slow mycelial growth were interrupted by periods of no growth (Fig. 1A). However, the growth pattern of ER-3 differed from the previously described stopper mutants in that ER-3 ceased to grow after one to several rounds of interrupted growth (Fig. IB). In many cases stopped periods were quite extensive, often exceeding 100 h. The stopping phenomenon in ER-3 was nearly the same as described for Abnormal-I and other stopper mutants in that the resumption of growth did not occur from the hyphal tips, but rather from an area several millimeters back from
DISTANCE
(ram)
0
- E ~ ~,~
I I
I I
.~. =. ~.~ ~ ~'~.~
i i i i i i i
~" ~ ~,~ ~• ~ ; ~
=" .~. ="
ilOwl~
m
IUUl
t
!
ill
~.
!
I
O"
IIiit
E.. DISTANCE
~ . ~
o
o . ~ =o"
~B ~o ='~..~ =-. a
.-,I
+5,~
i o~.~ 61
~
~
(ram)
~
~
§
20 the growth front. However, in m a n y experiments, E R - 3 displayed an apparent senescence, failing to resume growth even after 300 h (Fig. 1B). In E R - 3 , the senescence phenotype was often accompanied by the formation of droplets of a brown liquid on the mycelia near the growth frontier. Immediately following senescence, the medium distal to the growth front was found to darken, presumably due to the discharge of this brown liquid into the growth medium. This phenomenon was also seen when E R - 3 became senescent in liquid culture with a progressive darkening of the liquid medium shortly following cessation of growth. A heterokaryon was forced between E R - 3 [carrying a mutation for inositol auxotrophy (inl-)], and strain 2248 (a p a n - 2 mutant with normal growth and cytochrome content). Neither E R - 3 nor strain 2248 grew on minimal medium; however, the heterokaryon grew slowly at a rate significantly less than that of the wild type (Fig. 1A). The growth characteristics of the heterokaryon were much different than that of E R - 3 . In the heterokaryon, the stop-start pattern of linear growth was not observed in 500 h of growth. In addition, none o f the heterokaryons formed exhibited the senescence phenomenon. Also unlike the E R - 3 mutant, the heterokaryons produced conidia. A representative analysis o f conidia f r o m one heterokaryon presented in Table I and Fig. 1C showed two discrete groups: fast (wild type growth rate (approx. 3.8 m m / h ) , or slow (0.56 m m / h ) growers). However, the slow growing condial isolates grew at rates faster than the original E R - 3 mutant. Thus, the fast and slow growing isolates m a y represent the segregation of the normal and respiratory deficient mitochondria during the breakdown o f heteroplasmy. Also, the heterokaryon showed a wild type cytochrome spectrum. These data show that the heterokaryon behaved like the wild type strain. Thus, the suppressiveness in E R - 3 is apparently different from that described in other stopper mutants [2]. With the mutant determinant in the heterokaryon, it was of further interest to see if the heterokaryon could act as a protoperithecial parent in sexual crosses. If so, it might be possible to directly demonstrate maternal transmission to the progeny. Table II shows the result of such reciprocal crosses. When the heterokaryon acted as
TABLE I SEGREGATION OF NORMAL AND SLOW GROWING PHENOTYPES AMONG CONIDIAL ISOLATES FROM THE HETEROKARYONOF THE WILD TYPE AND STOPPER STRAINS Heterokaryon:
ER-3 (0.4) ~
Pan-2 (3.8) u
A In I- Pan [ - ]
A Inl*Pan-[+]
Growth phenotypes o f Pan-2 conidial isolates Normal Slow
Total
11 ( 3 . 8 ) a
75
64 (0.56) a
aGrowth rate (mm/h) indicated in parentheses.
21
TABLE II SEGREGATION OF NORMAL AND SLOW GROWING PHENOTYPES AMONG PROGENY OF THE CROSS BETWEEN WILD TYPE AND ER-3 (FIRST CROSS IN THIS TABLE) OR BETWEEN HETEROKARYONS CONTAINING THE STOPPER DETERMINANT (ER-3) AND THE WILD TYPE STRAIN (SECOND AND THIRD CROSSES IN THIS TABLE) Cross
Total
Ascospores
ascospores
germinated
Normal growers
SIow growers
240
94 (39.1 °70)
94
0
269
90 (33.5070)
85
5
224
24 (10.7070)
24
0
isolated 1. a Inl* Trp- [ + ]
x lnl- Trp ÷ [ - ] 2. a Inl* Pan* Trp- [ + ]
x A A 3. A A
InI- P a n ÷ Trp ÷ [ lnl* P a n - Trp ÷ [ + lnl" Pan" Trp ÷ [ I n l +P a n - Trp ÷ [ +
] + ] ] +
]
× a Inl +P a n ÷ Trp- [ + ]
The first member of each cross is the protoperithecial parent. Percent germination in parentheses.
the conidial parent (cross #2), the results were essentially identical to the cross involving ER-3 alone (cross * 1). In this experiment the tryptophan requiring mutant trp-3 (with wild type growth) was used as the perithecial parent and, as expected, almost all (94%) of the progeny were normal (wild type) growers. If, however, the heterokaryon was used as the perithecial parent, the progeny were again all wild type growers and none were slow growers. In all crosses performed with ER-3, the ascospore germination rate was exceedingly low. The ascospore germination was 33.5°70 when the heterokaryon was the conidial parent (cross #2) but only 10.7070 when it was the perithecial parent (cross #3). Thus, the result of sexual crosses involving ER-3 were similar to those reported for certain other stopper mutants [22].
Mitochondrial cytochrome content The senescence mutant ER-3 was found to possess abnormal levels of mitochondrial cytochromes (Fig. 2). The room temperature cytochrome spectrum of the mutant shows that the alpha cyt a + a 3 absorption peak at 610 nm is completely absent. However, this is in sharp contrast to the picture portrayed by the cytochrome soret bands. While the absorption maximum in the soret region is the same for both the wild type and ER-3, at 430 nm (not shown), the ratios of the gamma peak at 443 nm (cyt a 3) to that at 430 nm (cyt b) indicate that some component of cytochrome a is still present (Table III).
22
0.011
ER-3A
O Z 4( W
HK
Port °2A
500
550
600
650
WAVELENGTH (am)
Fig. 2. Oxidized versus reduced cytochrome difference spectra of the wild type (pan-2A), the senescence mutant (ER-3) and heterokaryon between them. Mitochondrial fractions were prepared, sonicated and solubilized with sodium deoxycholate. Sodium dithionite was added and the reduced spectra measured from 480 to 650 nm. Protein concentrations of preparations are: p a n - 2 A , 5.6 mg/ml; E R - 3 , 6.6 rag/ ml; heterokaryon 4.4 mg/ml.
TABLE II1 RELATIVE SPECIFIC CYTOCHROME MUTANT M I T O C H O N D R I A a Strain
CONTENTS OF WILD TYPE AND SENESCENCE
Cytochrome content ~
Soret
A 430 cyt b
cyt c
cyt a + a3
A46o RL3-SA ER-3A
0.0064 (0.0028) 0.0030 (0.0003)
0.0067 (0.0051) 0.0052 (0.0010)
0.0014 (0.0004) 0
0.015 0.018
=Cytochrome contents expressed as absorbance/mg per ml of mitochondrial protein. Values in parentheses are standard errors. Values for cytochromes b, c and a + a~ were derived as described in Methods from cytochrome alpha peaks. The soret band ratio for cyt a + a~ is derived from gamma peak values at 443 nm compared to absorbances at 460 nm.
23 The amounts of mitochondrial cytochromes from E R - 3 and the wild type strain RL3-8A presented in Table III indicate that the specific amounts of all three mitochondrial cytochromes are reduced in the mutant. In addition, the proportion of cytochrome b to cytochrome c is reduced by almost 40% as compared with that of the wild type. These data are consistent with the previously described stopper and poky mutants of N . crassa [6,22]. C y t o c h r o m e oxidase a n d respiration deficiency
In order to determine the functional relationship between the observed cytochrome deficiencies and the severely impaired growth rate of the mutant (0.398 _+ 0.032 m m / h ) versus the wild type (3.78 ___ 0.16 mm/h), two types of experiments were conducted. For the first, mitochondria were purified on sucrose gradients and cytochrome oxidase activity determined by means of a spectrophotometric assay. The results (Table IV) indicate that the specific activity of this enzyme in the mutant is only approximately 5% of that in the wild type. Table IV also portrays the results of the second series of experiments in which whole cell respiration rates were used as a measure of mitochondrial impairment. Whole cells were placed in fresh liquid growth medium in a Clark oxygen electrode and oxygen uptake was measured both with and without potassium cyanide present in the medium. The cyanide sensitive respiration rates appear to be identical for both the mutant and wild type, at approximately 12.5/amol of oxygen consumed/h per mg dry weight of mycelia. However, the total cell respiration rate of E R - 3 is 1.3 times greater than that of the wild type. Thus, the cyanide-resistant respiration represents a smaller fraction of total respiration in the mutant (17.5%) than in the wild type (22.5%).
TABLE IV CYTOCHROME OXIDASE AND WHOLE CELL RESPIRATION RATES OF THE WILD TYPE AND SENESCENCEMUTANT' Strain
RL3-8A ER-3A
Cytochrome c oxidase
61.8 (5.3) 3.1 (0.4)
Whole cell respiration Total
KCN resistant
54.41 (9.06) 72.66 (7.45)
12.42 (3.43) 12.72 (2.99)
•Cytochrome c oxidase values are expressed as a change in absorbance/min per milligram of mitochondrial protein and are the averageof two trials. Whilecell respiration rates are expressed as micromolesof oxygen consumed/h per mg of dry weight of cells. Values for RL3-8A are the averages from three trials and those for ER-3A are averagesof four trials. Valuesin parentheses are standard errors.
24
Effect o f respiratory inhibitors Stopper mutants are characterized by the unique stop-start growth phenotype displayed by mycelia grown on solid media. When such stopper mutants are grown in race tubes [14] and their linear growth measured over the course of time, the stopstart pattern is readily apparent (Fig. 2). The processes responsible for this abnormal growth pattern are not fully understood, however, it was of considerable interest to use this trait as a measure of respiratory sufficiency in growing cells. It was hoped that it would be possible to provide evidence for a biochemical defect in vivo by growing the mutant in the presence of respiratory inhibitors and examining the effect on the mutant growth pattern. The concentrations of inhibitors used in these studies were taken f r o m previously published studies of Neurospora grown in liquid culture [19--20]. It was also desired that growth of the wild type strain in the presence of such inhibitors would induce respiratory deficient or stopper phenocopies. The generation of such phenocopies could provide additional information concerning the identification of the stopper biochemical defect in vivo. The results of these experiments are shown in Fig. 3. The typical growth pattern for wild type is that of the control (Fig. 3A). When the wild type was grown in the presence of 1 m M cyanide, its initial lag phase was somewhat lengthened as compared to that in the absence of inhibitor. However, after 2 days a normal growth rate was resumed (Fig. 3A). Growth in the presence of salicyl hydroxamic acid (SHAM), an inhibitor of the Neurospora cyanide-resistant alternate oxidase, was initially comparable to that in the absence of inhibitor, but progressively slowed until it attained a rate approximately one fourth that of the control. Growth in the presence of cyanide and S H A M together produced a pattern which mirrored that obtained in the presence of S H A M alone. However, the additional effect of cyanide was again evident as a lag during the initial 24 h of growth (Fig. 3A). The effect of sodium azide on the growth of the wild type was even more pronounced. Wild type mycelia grown in the presence of azide together with either cyanide or S H A M grew at a rate comparable to that of the mutant (ER-3). However, in the presence of azide, cyanide had a much more inhibitory effect on growth than S H A M (Fig. 3A). Figure 3B shows the results typically obtained when ER-3 is grown in the presence of the same inhibitors (i.e., cyanide, S H A M and sodium azide). As with the wild type, growth of ER-3 in the presence of 1 mM cyanide is slowed as compared with the rate of growth in the absence o f the inhibitor. ER-3 did not grow in the presence of either S H A M or sodium azide (Fig. 3B). The second set of inhibition studies involved antimycin A, oligomycin and the uncoupler 2,4-dinitrophenol (Figs. 3C and 3D). The only apparent effect of those compounds on the wild type was a somewhat longer lag in initial growth and a slightly reduced rate o f growth compared to that in the absence of inhibitors (compare Figs. 3A and 3C). However, the mutant was unable to grow beyond germination in the presence of the uncoupler (Fig. 3D).
25
50O
400
400
300
~3OO
200
¢:
2OO
I00 I00
IOO
200
I
300
I00
200 TIME
TIME (hours) SO0
5001
400
40C
300
"~300
300
(hours)
z
