Current Genetics
Current Genetics (1981)4:29-36
© Springer-Verlag 1981
Isolation and Characterization of Yeast Mitochondrial Mutants Defective in Spore Germination Andreas Hartig 1, Ren6e Schroeder2, Eva Mucke, and Michael Breitenbach Institut ftir AUgemeineBiochemieand Ludwig Boltzmann-Forschungsstellefiir Biochemie,WShringerstrasse 38, A-1090 Wien, Austria
Summary.This paper describes a new type of mitochondrial mutation. During germination of ascospores the mutants are blocked at the first budding stage and consequently die. However, vegetative growth on nonfermentable carbon sources and respiration are close to normal. The spores of the mutants which (like the wild type) contain very low amounts of mitochondrial cytochromes, do not synthesize cytochromes b and aa 3 during germination. The mutants show a pleiotropic phenotype during the vegetative phase: they lack carbon catabolite repression of cytochromes on media containing 10% glucose. We discuss here the hypothesis that the mutation is located in a regulatory region on the mitochondrial genome which is needed for the reinitiation of mitochondrial genetic activity during germination of ascospores. Key words: Germination of ascospores - Mutation in mtDNA - Mitochondrial differentation
Introduction
The biogenesis of mitochondria during cell differentiation and the contribution which mitochondria make to
Offprint requests to: MichaelBreitenbach 1 Present address: National Institutes of Health, NINCDS
Bldg. 36, 3DO2, Bethesda, MD 20205, USA 2 Present address: Genetisches Institut der Universit~it, Maria
Wardstrasse la, Miinchen, BRD
this process has attracted comparatively little attention up to now. It seems to be clear that mitochondrial differentiation does occur in animal (Pollak and Sutton 1980) and in yeast cells (Brewer and Fangman 1980). Yeast cells may undergo three different morphogenetic differentiation processes, depending on their genetic constitution and the physiological stimuli provided by the medium. These are mating, sporulation, and germination. In cells devoid of mitochondrial DNA mating is possible and sporulation is blocked completely. Therefore, the necessity of mitochondriaUy encoded functions is ruled out for the mating process, but left open for sporulation and germination. Inhiqgitor studies show that mitochondrial transcription (Newlon and Hall 1978) and translation (Puglisi and Zennaro 1971) is needed during yeast sporulation. Similar data concerning the germination process in yeast have been presented (Rousseau and Halvorson 1973a) and the reinitiation of mitochondrial functions during germination has been studied using B o t r y o d p l o d i a t h e o b r o m a e spores (Brambl 1977; Brambl and Josephson 1977). Our approach to this interesting question was to study the sporulation properties of respiratory deficient mitochondrial mutations in order to identify genetic loci that are specifically needed during sporulation (Hartig and Breitenbach 1980). This led us to investigate mitochondrial transcription and translation during yeast sporulation and germination (Schroeder and Breitenbach 1981a). In the present communication we will present a new type of mitochondrial mutation which further corroborates the notion of differentiation specific mitochondrial genes. The mutants grow on non-fermentable carbon sources and respire almost normally, but are blocked during germination of the spores. We shall describe the mutant selection system, the mode of inheritance and the physiological characterization of the mutants. 0172-8083/81]0004]0029/$01.60
A. Hartig et al.: Germination-specific Mitochondrial Mutants
30 Table 1. Strain
Genotype
Origin
nuclear AP-3
a a
mitochondrlal
adel +
ade2-x ural ade2-y +
his7
+
+
lys 2 tyrl +
AP-3 rec F 101-1 F 102-2 JC8-AA1 V-13, 17, 19, 22 V-17 rec. V-17/5 V-319 V-320
Remarks
+
gall +
like AP-3 a, leu2 a, leu2 a, karl, leul like AP-3 like AP-3 a, ade2 F101-1 x JC8-AA1 F101-1 x JC8-AA1
[p+]
A. Hopper
[o + ] [o ° ] [po ] [pO] [p+, g e r - ] [p+, g e t - ] [p+, ger-] [p+, g e r - ] [p+, ger +]
our laboratory our laboratory our laboratory G. Fink our laboratory our laboratory our laboratory our laboratory our laboratory
+
chx2 can1
Materials a n d M e t h o d s
Yeast Strains. The strains used, their genotypes and origins are summarized in Table 1. Strain V-319 was constructed by cytoduction as described under Genetic Analysis in the results section. Strain V-320 is the isogenic wild type corresponding to V-319. It was constructed in a similar way, but starting from a haploid a strain derived from AP-3 by tetrad analysis. Chemicals. Glucose, sucrose, potassium acetate and mercaptoethanol were of p.A. grade from Merck (Darmstadt, GFR). Agar, yeast nitrogen base without amino acids, peptone and yeast extract were from Difco (Detroit, USA). Amino acids, adenine, uracil, cycloheximide, eanavanine and calf thymus DNA were from Sigma (St. Louis, USA). Glusulase was from ENDO Laboratories (Garden City, NY, USA). Percoll was from Pharmacia (Uppsala, Sweden). Dlamidinophenyl indole was a gift from Dr. O. Dann (Erlangen, GFR). All other chemicals were of reagent grade. Media; Growth, Sporulation and Germination Conditions. The yeast strains were kept on YPD agar slants (containing 2% agar 2% glucose, 2% peptone and 1% yeast extract) at 4 °C. Standard sporulation experiments were performed as follows: Diploid strains were grown overnight at 30 °C in well aerated liquid medium containing 0.5% glucose and 1% yeast extract. The stationary cultures were diluted 1 : 1 with fresh medium containing 0.3% glucose and 1% yeast extract and grown for an additional 2 h. During this period glucose was used up completely and the cell densities nearly doubled. The ceils were now harvested and washed three times, and then resuspended in 1% potassium acetate at a density of 5 x 107 cells per ml. After 48 h sporulation was complete under these conditions. The experiments were performed either in Erlenmeyer flasks in rotary shakers (up to 1 1 of total volume) or in a New Brunswick type Microferm fermenter (10 1 of total volume). For the germination experiments, the spores were suspended in vegetative growth medium (1% glucose, 0.3% peptone, 0.8% yeast extract) containing 0.1% Triton X-100 and shaken at 200 rev/min at 30 °C.
mitotic recombinant in ade-2 isolated by F. Fel~l described in (Hartig and Breitenbach 1980) described in (Conde and Fink 1976) mitotic recombinant in ade-2 isomitochondrial with V-17 isomitochondrial with AP-3
For the glucose repression and derepression experiments the following media were used: 10% glucose, 1% yeast extract (YD) or 3% glycerol, 1% yeast extract (YG), respectively. For the purpose of tetrad analysis small scale sporulation experiments were performed by growing the diploid strains on YPD agar and replica plating on solid sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose and 2% agar). YPG (3% glycerol, 2% peptone, 1% yeast extract, 2% agar) was used for the detection for respiratory deficient colonies after mutagenesis. SD (0.67% yeast nitrogen base without amino acids, 2% glucose, 2% agar) was used for the isolation of diploid single colonies after crosses. SD+ leucine (30 ppm) was used for the isolation of cytoductants after crosses with strain JC8-AA1. Selection medium was SD+ 20 ppm adenine, 20 ppm uracil, 20 ppm histidine, 30 ppm lysine, 30 ppm tyrosine, 20 ppm cycloheximide and 80 ppm canavanine sulfate. This medium served for the discrimination of diploids derived from strain AP-3 (sensitive to cycloheximide plus canavanine) from haploids resistant to cycloheximide plus canavanine. All media were used with or without the addition of 2% agar.
Cell densities and percentage of asci were counted in a modified Neubauer hemocytometer. Mutagenesis and Mutant Selection. Strain AP-3 was grown to a ceil density of I x 10 "/ cells/ml in 10% liquid YPD (10% glucose, 2% peptone, 1% yeast extract). At this stage MnC12 solution was added to a final concentration of 1 mM and the culture kept at 30 °C without shaking for one day. Under these conditions stxain AP-3 grew to about 1 x 10 s cells/ml- The culture was diluted and plated onto 100 YPD plates (approximately 200 colonies per plate). After two days colonies showed up and were replica plated on YPG and sporulation agar. After another two days the sporulation plates were replica plated onto the selection medium. Two days later the replicas of wild type colonies exhibited growth on the selective medium, indicating the presence of living haploids (Fig. 1). Colonies growing on YPG, but not on selective medium were picked, subcloned four times and further analyzed genetically. The expected phenotype of these clones was respiratory sufficient and sporulation or germination deficient.
A. Hartig et al.: Germination-specific Mitochondrial Mutants
~
31 All recordings of cytochrome spectra were performed with strains not carrying the homozygous ade-2 markers. These were either: mitotic recombinants of the two heteroallelic ade-2 markers or in one case strain V-320. This was necessary, because the red pigment produced by ade-2 strains obscures the cytochrome spectra.
PD
YPG ~ o o medium Genetic and Physical Mapping Procedures. The techniques used have been described elsewhere (Schroeder and Breitenbach 1981a).
@
setection medium
Results
Isolation o f the Mutants Fig. 1. Mutant selection scheme. Full circles: growing colonies, open circles: nongrowing colonies. The arrow indicates a colony that is respiratory sufficient (growing on YPG), but differentiation deficient (nongrowing on selection medium)
Fluorescent Staining. The DNA-specific fluorescent stain diamidinophenyl indole was used to visualize nuclear and mitoehondrial DNA yeast ascospores. The method used was that of Williamson and Fennell (1975). Preparation o f Yeast Aseospores. Yeast cells were sporulated in 1% potassium acetate as described above, harvested and treated with mercaptoethanol and glusulase to remove ascus wails. The spore tetrads were then shaken gently with glass beads in 0.5% Triton X-100 to obtain single spores and layered on top of Percoil gradients (75-90% Percoll, 0.25 M saccharose). After centrifugation for 1 h at 10,000 rpm (Sorvall SS-34 rotor) a spore pellet had separated from the remaining vegetative cells, ascus wails and debris. Centrifugation was repeated once. The spores which were better than 99.9% pure were kept in 0.5% Triton X-100 at 4 °C. Wild type spores remained completely viable for at least a year. Details of the procedure will be published elsewhere. Light Microscopy. Germinating yeast spores were photographed using a Reichert Diavar phase contrast microscope equipped with an automatic camera. Measurement o f the respiratory activity o f the cells and micromanipulation were performed as described earlier (Hartig and Breitenbach 1980). DNA determination was performed as described by Schroeder and Breitenbach (1981b). Recording Cytoehrome Spectra. a) Vegetative cells were grown overnight in YG to a cell density of 5 x 107 cells/ml, diluted with fresh YG to a cell density of 1 x 107 cells/ml, grown for another 8 h and harvested at a cell density of 3 to 4 x 107 cells/ml (derepressed cells). Alternatively, the overnight cultures were diluted with YD (10% glucose) to a cell density of 2 x 107 cells/ml, grown for another 8 h and harvested at a ceil density of 4 x 107 cells/ml. The cells were treated with an excess of Na2S204 in suspension, quickly centrifuged, the pellet applied to the window of a home-made cuvette and frozen in liquid nitrogen. Spectra were recorded against several layers of Parafilm in a Beckmann LS 24 recording spectrophotometer, b) Spores were isolated on Percoll gradients as described above and treated in the same way as vegetative cells for the recording of the cytochrome spectra.
Out of 20,000 primary clones, we isolated 22 colonies that grew normally on glycerol b u t failed to produce living hyploids. Four of them were sporulation deficient (spo-). All four had gained the ability to mate with appropriate tester strains. We assumed that these were mutations in the mating type locus (or epistatic over the mating type locus) and excluded them from further analysis. The remaining 18 mutants produced asci in high yield b u t b y mass spore analysis on selective medium (Fig. 1) no germination was observed. Upon subcloning and analysis of individual spores four of the mutants (V-13, 17, 19, 22) showed a stable phenotype of germination deficiency, i.e. the spores did not form colonies on YPDagar.
Genetic Analysis For the purpose of genetic analysis it was necessary to isolate haploid clones carrying the mutation. Two of the four mutants mentioned (V-17 and V-19) were not totally germination deficient b u t produced living haploids at a low frequency (1 in 107 to 1 in 106) when spores were plated on selective medium. Some of the haploids were tested for mutant or wild type phenotype b y crossing them with rho ° - mating partners, isolating and sporulating the diploid clones and testing spore tetrads for their ability to form colonies on YPD-agar. The spores again showed the stable phenotype of germination deficiency. Some, however, were wild type with respect to germination. The theoretical implications of this behaviour will be part of the discussion. Haploid V-17/5 was used for further genetic analysis. It was crossed with strain JC8-AA1 (rho °) and haploids were isolated from the cross that carried the nuclear genome of JC8-AA1, b u t the mitochondrial genome of V-17/5 (Conde and Fink 1976). One such strain was crossed with strain F-101-1; the spores of the resulting diploid (V-319) again were germination deficient. This cytoduction experiment with strain JC8-AA1 revealed
32
A. Hartig et al.: Germination-specificMitochondrialMutants
•106c eLtS/m l
n moles Oz/rninJO6ceL[ s
,106cetl.S/m L 100 40
Fig. 2a. Growth on YPD (1%glucose) medium. Circles: wild type AP-3, triangles: mutant V-17, b Growth on YPG (3%glycerol)medium, c Respiration during growth on YPG (3% glyc-
50 20
12
24 h
12
b
24
48
h
c
c
n mol.es 02/min,106c ett s
°/oasci
30 100Olo
] \
/
• 20
12
A
h
48
erol)
Phenotypie Characterization of the Mutants
550 600 wavelength [nm]
600
Fig. 3AID. Low temperature cytochrome spectra of vegetative cells. A Wild type AP-3 rec. on YG (3% glycerol). B Mutant V-17 rec. on YG (3% glycerol. C Wild type AP-3 rec. on YD (10% glucose). D Mutant V-17 rec. on YD (10% glucose)
increase
24
that the germination deficiency of strain V-17 was inherited by a cytoplasmic genetic element. By a series of mitochondrial crosses a single genetic factor on mitochondrial DNA could be shown to be responsible for the germination deficieny. Its location is between the genes cob and oli2 (Breitenbach et al. in preparation).
E-l,0
550
12
~mo/.es 02/, . .^6
24
36 h
.
60 100% DNA e increase
~
40
20
12
24
36 h
Fig. 4. A Wild type AP-3 during sporulation. Triangles: Respiration, Circles: premeiotic DNA synthesis, Squares: percent asci, B Mutant V-17 during sporulation. Symbols as in Fig. 4. A
a) Vegetative Growth. As shown by the data presented in Fig. 2a and 2b growth of mutant and wild type on media containing 1% glucose or 3% glycerol as a carbon source was similar. Oxygen uptake of the cells during growth on glycerol medium was also nearly identical in mutant and wild type (Fig. 2c). The cytochrome spectra of mutant and wild type after growth on 10% glucose and 3% glycerol, respectively, were drastically different (Fig. 3). The wild type showed the well known pattern of glucose repression: On ] 0% glucose the characteristic absorption bands of cytochromes aa3, b, c 1 and c are 5 to 10 times less intense than on 3% glycerol. The mutant spectrum on 10% glucose was nearly as intense as the wild type spectrum on 3% glycerol, i.e. the mutant lacked carbon catabolite repression of cytochromes. On the other hand, the mutant spectrum on glycerol was nearly normal. The possible correlation of the lack of carbon catabolite repression with the germination deficiency will be discussed below. b) Sporulation. Our first idea was to look for biochemical defects during the sporulation phase, reasoning that the absence of some of the substances normally present in spores could cause the germination deficiency. However, most of the biochemical and cytological criteria of sporulation that we tested in the mutant appeared to be quite normal: Respiration, DNA synthesis and the percentage of mature asci during sporulation experiments reveal no significant differences between mutant and wild type (Fig. 4a, b). The morphology of the mutant spores as
A. Hartig et aL: Germination-specificMitochondrial Mutants
33 of an ascus. In some cases "blobs" representing mitochondrial DNA (Wiltiamson and Fennell 1975) could be seen in the asci, inside as well as outside the spores. Also in this respect we could not find any difference between wild type and mutant. The mutant spores seemed to contain mitochondrial DNA. The only difference between mutant and wild type spores which we have found so far is their behavior on Percoll density gradients. In the stepgradients used (75-90% Percoll) the wild type spores sedimented to the bottom of the centrifuge tube. Under the same conditions the mutant spores banded at the boundary between 85% and 90% PercoU. This was true for spores of both the strains V-17 and the isomitochondrial but nuclearly unrelated V-319.
Fig. 5 a and b. Mature asci stained with diamidinophenylindole and viewed in the fluorescence microscope, a Wild type AP-3. b Mutant V-17 judged by phase contrast microscopy also seemed to be quite normal. Figures 5a and 5b show mature asci of wild type and mutant which have been stained with diamidinophenyl indole. In those asci which were oriented properly four spore nuclei with about equal fluorescence could be seen. This means that nuclear DNA probably segregates normally to the four spores
c) Germination. As observed by phase contrast microscopy, wild type spores during germination on YPD (1% glucose) behaved as described in the literature (Rousseau 1972). The spores went through the following stages: a) "Germination proper", i.e. phase darkness and some swelling of the spores (30 rain to 1 h after suspension in YPD), b) "outgrowth", i.e. elongation of the spores (1-3 h in YPD), c) "budding". The first buds appeared around 4 h after the start of the experiment. Electron micrographs showed that the first two or three budding cycles differ from purely vegetative cycles in that the cell walls were considerably thicker than normal vegetative cells walls (data not shown here). This means that germination specific processes probably occur for upt to 10 h in germination medium. The formation of zygotes appears only after the first b udding. Mutant strain V-17 went through the stages of phase darkness and elongation of the spores (however at a slower rate than the wild type) but was arrested during the first budding cycle. Some of the spores did not bud; however, they were enlarged to a size greater than normal vegetative cells. At 6.5 h many spores had already lysed.
ng ONA/106 spores
n m°[ ee 02/rnin, 106spores
6C
,/
/
.9
40
.7
J
.5
2C
.3
.2 .1 a
.1 2
4
6
8
10
12
26 h
b
2
4
6
8
10 h
C
2
4
6
8
1~3 h
Fig. 6. a Growth of spores under conditions of germination. Optical density was measured at 546 nm. Triangles: wild type, circles: mutant V-17. b DNA synthesis under conditions of germination. Triangles: wild type AP-3, circles: mutant V-17, e Respiration of spores under conditions of germination. Triangles: wild type AP-3, circles: mutant V-17
34
A. Hartig et al.: Germination-specific Mitochondrial Mutants
Fig. 7. a Low temperature cytochrome spectra of spores of the wild type strain V-320 during germination. The numbers next to the spectra indicate hours in germination medium, b Low temperature cytoehrome spectra of spores of the mutant strain V-17 rec. during germination. Symbols as in Fig. 7a 500
600
nm
560
600
nm
The germination deficient spores could not be rescued by mating them with strains of a or ~ mating type. This was checked by micromanipulating mutant spores and placing one or two vegetative haploid cells of the same mating type next to every member of a tetrad. In no case did the growing colonies contain diploid cells. As can be seen in Fig. 6b, the wild type grows with a doubling time of about 2 h from the time of the first budding, whereas the mutant stops growing around 8 h By this time its cells number and biomass have less than doubled. DNA synthesis in germinating mutant spores also reflects this situation. Figure 6a shows that the mutant spores approximately doubled their DNA content during the first 6 - 8 h of the germination experiment, then stopped synthesizing DNA. Respiration of germinating spores (Fig. 6c) started during the outgrowth of wild type spores. The mutant spores reached only a low level of respiration, corresponding approximately to one tenth of the maximal respiration attainable by strain AP-3. The time course of the synthesis of spectroscopically visible cytochromes during the germination of wild type spores is shown in Fig. 7a. Mature spores contain a very small amount of cytochromes. During germination cytochromes c, b, and aa 3 are synthesized at different times. Cytochrome c appears after 4 - 6 h, b after 6 - 8 h and aa 3 only after 10-12 h. Figure 7b shows that the mutant spores do synthesize cytochrome c at the normal rate, but they do not synthesize cytochromes b and aa 3 at all over a time course of 24 h. In other words, mutation V-17 has a pleiotropic effect on the production of both cytochromes b and aa3.
Discussion The strain used for the mutant selection procedure as described here, is a normal diploid a/c~ strain. This means that only dominant nuclear mutations or cytoplasmic mutations can be detected. The procedure does not discriminate between sporulation deficient and germination deficient mutants. As has been described in this paper, we have predominantly isolated mutants of a single phenotype, namely respiratory competent mutants which are germination deficient and as a pleiotropic effect are deficient in carbon catabolite repression of cytochromes during vegetative growth. One of the mutants has been mapped on the mitochondrial genome. Nuclear mutations have also been isolated (Baranowska et al. 1977), but were not investigated for the purpose of this paper. Based on the results of our previous work on the role of the mitochondrial genome in yeast sporulation (Hartig and Breitenbach 1980), we also expected to find mitochondrial mutations which are respiratory competent but sporulation deficient. Such mutants have not been found up to date. Genetic characterization of the germination deficient mutants was possible due to the fact that with a frequency of about 10 .7 spores were found to grow out. Their progeny was composed of a majority of mutant cells still carrying the ger- mutation, and a minority of get + cells, which probably are revertants. We explain their occurrence as follows: In one cell, and even in one sPore a multiplicity of mitochondrial genome exists. Careful morphometric work (Brewer and Fangman 1980) showed that spores contain probably 1 0 - 1 4 mito-
A. Hartig et al.: Germination-specificMitochondrialMutants chondrial genomes. In those strains that revert at a low frequency, the presence of only one or two wild type mitochondrial genomes may suffice to enable the spore to outgrow normally. During growth of the resulting haploid colony the ger+ and ger- mitochondrial genomes segregated to different cells lines. The method was only applicable for one of the mutant strains. The differentiation of mitochondria during germination of yeast spores is one of the most striking results of the present work. The cytochrome spectra (Figs. 7a, b) and the complete absence of respiration in resting spores indicate that the mitochondria of the spores are in an extremely repressed state. Electron microscopic evidence further corroborates this fact. Sando et al. (1980) found that the mitochondria of spores contain little internal structure (cristae), in fact they have been termed "promitochondria-like" in the older literature (Hashimoto et al. 1958). Brewer and Fangman (1980) have shown by serial sectioning 4 different asci, that the spores in most cases contain one large mitochondrion that is described as "fermentation4ike". During germination the ultrastructural features of the mitochondria slowly change to "normal" (Kreger-Van Rij 1978). Our results show that the three cytochromes c, b, and aa3 are synthesized at different rates. The nuclear encoded cytochrome c appears during the "outgrowth" phase, but the (partly) mitochondrially encoded cytochromes b and aa 3 appear at six and after 10 h in germination medium, respectively. These results are in excellent agreement with the work of Brambl and Josephson (1977) and Brambl (1977), wo have measured cytochrome spectra during the germination of Botryodiplodia theobromae spores. They found virtually the same kinetics for the appearance of cytochromes c, b and aa 3 in those spores. This could mean that the extreme repression of mitochondria and the slow and asynchronous reappearance of mitochondrial cytochromes during germination is a gerneral phenomenon in fungal spores. In the case of Saccharomyces cerevisiae spores, we do not know at present whether de novo synthesis of cytochromes or some other process (modification, assembly in the inner mitochondrial membrane) is responsible for the appearance of the visible cytochromes. In the case of Botryodiplodia spores it has been shown that the novo mitochondrial protein synthesis is indeed necessary for the reappearance of cytochromes b and aa3. The germination deficient mutant described here is blocked at the time of the first budding cycle, which is a rather late stage of germination. It produces nearly normal amounts of cytochrome c, but no cytochromes b and aa 3 during germination. This is in good agreement with the fact that the partly mitochondrially synthesized cytochromes b and aa3 also appear later in germination and that fermentatively usable carbon sources are absolutely needed for the first few hours of germination
35 (Banerjee 1971, and our own unpublished results). One hypothesis which would account for this fact, is that the mutant is unable to reactivate the mitochondrial protein synthesizing system during germination, but this hypothesis has to be tested experimentally. The late manifestation in germination of mutant V-17 points to the fact that germination-specific processes occur for up 10-12 h after suspension of the spores in germination medium. Similar conclusions have been reached in the literature (Rousseau and Halvorson 1973 a and b; Rousseau 1972; Savarese 1974; Kreger van Rij 1978) based mainly on electron microscopic observations: Three stages ("germination proper", "outgrowth", and "budding") can be distinguished. The outer spore wall, which is very osmiophilic, ruptures during "outgrowth", but the new wall synthesized during the first two to three budding cycles is similar to the inner wall of spores and only gradually changes to a vegetative cell wall. What is the role of the yeast mitochondrial genome in germination and how does mutation V-17 interfere? Concerning spomlation there is now evidence that specific mitochondriai genes and mitochondrial protein synthesis are required for the normal completion of the process (Puglisi and Zennaro 1971; Newlon and Hall 1978; Hartig and Breitenbach 1980). Sporulation-and germination-specific products of mitochondrial transcription and translation have been found (Schroeder and Breitenbach 1981a). In contradiction to that, Tingle et al. (1974) reported that spores which are devoid of mitochondrial DNA can grow out and form haploid colonies. However, some objections have been raised against their conclusions (Newlon and Hall 1978; Hartig and Breitenbach 1980). We believe that these experiments do not rigorously exclude the presence and expression of mitochondrial genes during sporulation and germination. Evidence favoring a specific participation of the mitochondrial genome in germination has been scare up to now. Rousseau and Halvorson (1973a) found that erythromycin is an inhibitor (to some extent) of the germination o f yeast spores, indicating that mitochondrial protein synthesis may be necessary for germination. The results described in the present paper point to a specific role of a mitochondrial gene in germination. A mutant in that gene, which blocks germination during the formation of the first bud, leaves vegetative growth nearly unaffected. Genetic and physical mapping locates the mutational site to within 2,000-3,000 bp in the region between the genes cob and oli2 on the mitochondrial genome (Breitenbach et al. in preparation). No sequences and no other genetic data are available at present from this region. Therefore, it is not possible to draw any conclusion as to the expression of this gene. It may be transcribed and translated, only transcribed, or it may be a nontranscribed regulatory DNA sequence. The pleiotrophic, "regulatory" character of the mutation is documented by its
36 phenotype: during vegetative growth, the glucose repression of cytochromes is missing, the spores exhibit a density different from wild type o n Percoll gradients, and during germination appearance of cytochromes b and aa 3 is blocked.. We believe that it is the repressed state of spore mitochondria that necessitates a specific mitochondrial gene for the reactivation of the mitochondrial genetic system during germination and that m u t a t i o n V-17 confers a defect in such a regulatory gene.
Acknowledgement: The authors wish to express their gratitude to Dr. R. J. Schweyen and Dr. H. Lehrach for their help and encouragement during the course of this work and for many fruitful discussions, to Dr. W. Schnedl for his help with the fluorescent staining of DNA and to Prof. O. Hoffmann-Ostenhof for critically reading the manuscript. This work was supported by the Ludwig Boltzmann-Gesellschaft and by a grant from the Jubiliiumsfonds der ~sterreichischen Nationalbank, No. 1106.
References Banerjee M (1971) Ph.D. thesis, McMaster University, Hamilton, Ontario Baranowska H, Ejchart A, Putrament A (1977) Mutat Res 42: 343-348 Brambl (1977) Arch Biochem Biophys 182:273-281 Brambl R, Josephson M (1977) J Bacteriol 129:291-297 Brewer BJ, Fangman WL (1980) Proc Natl Acad Sci USA 77: 5380-5384 Conde J, Fink GR (1976) Proc Natl Acad Sci USA 73:36513655 Diala ES, Wilkie D (1977) In: Bandlow W, Schweyen RJ, Wolf K,
A. Hartig et al.: Germination-specific Mitochondrial Mutants Kaudewitz F (eds) Mitochondria. De Gruyter, Berlin New York, pp 563-570 Hartig A, Breitenbach M (1980) Curt Genet 1:97-102. Hashimoto T, Conti SF, Naylor HB (1958)J Baeterio176:406416 Kreger-Van Rij NJW ( 1978) Arch Microbio1117: 73 -77 Newlon MC, Hall BD (1978) Gen Genet 165:113-114 Pollak JK, Sutton R (1980) Trends in Biochem Sci 5:23-27 Puglisi PP, Zennaro E (1971) Experientia 27:963-964 Putrament A, Baranowska H, Prazmo W (1973) Mol Gen Genet 126:357-366 Rousseau P (1972) J Bacteriol 1.09:1232-1238 Rousseau P, Halvorson HO (1973a) Can J Microbiol 19:13111318 Rousseau P, Halvorson HO (1973b) J Baeteriol 113:12891295 Rousseau P, Halvorson HO (1973c) Can J Microbiol 19:547555 Sando N, Oguchi T, Nagano M, Osumi M (1980) J Gen Appl Microbiol 26:403-412 Savarese JJ (1974) Can J Microbiol 20:1517-1522 Schroeder R, Breitenbach M (1981a) In: Levinson HS, Sonenshein AL, Tipper DJ (eds) Sporulation and Germination (in press) Proceedings of the 8th Int. Spores Conference. A.S.M. Publications Schroeder R, Breitenbach M (1981b) J Bacteriol 146:775-783 Tingle MA, Kiienzi MT, Halvorson HO (1974) J Bacteriol 117: 89-93 Williamson DH, Fennell DJ (1975) In: Prescott DM (ed)Methods in cell biology, Vol. XII Academic Press, New York,pp. 335352
Communicated b y R.J. Schweyen Received May 15/July 20, 1981
Current Genetics (1981)4:29-36
© Springer-Verlag 1981
Isolation and Characterization of Yeast Mitochondrial Mutants Defective in Spore Germination Andreas Hartig 1, Ren6e Schroeder2, Eva Mucke, and Michael Breitenbach Institut ftir AUgemeineBiochemieand Ludwig Boltzmann-Forschungsstellefiir Biochemie,WShringerstrasse 38, A-1090 Wien, Austria
Summary.This paper describes a new type of mitochondrial mutation. During germination of ascospores the mutants are blocked at the first budding stage and consequently die. However, vegetative growth on nonfermentable carbon sources and respiration are close to normal. The spores of the mutants which (like the wild type) contain very low amounts of mitochondrial cytochromes, do not synthesize cytochromes b and aa 3 during germination. The mutants show a pleiotropic phenotype during the vegetative phase: they lack carbon catabolite repression of cytochromes on media containing 10% glucose. We discuss here the hypothesis that the mutation is located in a regulatory region on the mitochondrial genome which is needed for the reinitiation of mitochondrial genetic activity during germination of ascospores. Key words: Germination of ascospores - Mutation in mtDNA - Mitochondrial differentation
Introduction
The biogenesis of mitochondria during cell differentiation and the contribution which mitochondria make to
Offprint requests to: MichaelBreitenbach 1 Present address: National Institutes of Health, NINCDS
Bldg. 36, 3DO2, Bethesda, MD 20205, USA 2 Present address: Genetisches Institut der Universit~it, Maria
Wardstrasse la, Miinchen, BRD
this process has attracted comparatively little attention up to now. It seems to be clear that mitochondrial differentiation does occur in animal (Pollak and Sutton 1980) and in yeast cells (Brewer and Fangman 1980). Yeast cells may undergo three different morphogenetic differentiation processes, depending on their genetic constitution and the physiological stimuli provided by the medium. These are mating, sporulation, and germination. In cells devoid of mitochondrial DNA mating is possible and sporulation is blocked completely. Therefore, the necessity of mitochondriaUy encoded functions is ruled out for the mating process, but left open for sporulation and germination. Inhiqgitor studies show that mitochondrial transcription (Newlon and Hall 1978) and translation (Puglisi and Zennaro 1971) is needed during yeast sporulation. Similar data concerning the germination process in yeast have been presented (Rousseau and Halvorson 1973a) and the reinitiation of mitochondrial functions during germination has been studied using B o t r y o d p l o d i a t h e o b r o m a e spores (Brambl 1977; Brambl and Josephson 1977). Our approach to this interesting question was to study the sporulation properties of respiratory deficient mitochondrial mutations in order to identify genetic loci that are specifically needed during sporulation (Hartig and Breitenbach 1980). This led us to investigate mitochondrial transcription and translation during yeast sporulation and germination (Schroeder and Breitenbach 1981a). In the present communication we will present a new type of mitochondrial mutation which further corroborates the notion of differentiation specific mitochondrial genes. The mutants grow on non-fermentable carbon sources and respire almost normally, but are blocked during germination of the spores. We shall describe the mutant selection system, the mode of inheritance and the physiological characterization of the mutants. 0172-8083/81]0004]0029/$01.60
A. Hartig et al.: Germination-specific Mitochondrial Mutants
30 Table 1. Strain
Genotype
Origin
nuclear AP-3
a a
mitochondrlal
adel +
ade2-x ural ade2-y +
his7
+
+
lys 2 tyrl +
AP-3 rec F 101-1 F 102-2 JC8-AA1 V-13, 17, 19, 22 V-17 rec. V-17/5 V-319 V-320
Remarks
+
gall +
like AP-3 a, leu2 a, leu2 a, karl, leul like AP-3 like AP-3 a, ade2 F101-1 x JC8-AA1 F101-1 x JC8-AA1
[p+]
A. Hopper
[o + ] [o ° ] [po ] [pO] [p+, g e r - ] [p+, g e t - ] [p+, ger-] [p+, g e r - ] [p+, ger +]
our laboratory our laboratory our laboratory G. Fink our laboratory our laboratory our laboratory our laboratory our laboratory
+
chx2 can1
Materials a n d M e t h o d s
Yeast Strains. The strains used, their genotypes and origins are summarized in Table 1. Strain V-319 was constructed by cytoduction as described under Genetic Analysis in the results section. Strain V-320 is the isogenic wild type corresponding to V-319. It was constructed in a similar way, but starting from a haploid a strain derived from AP-3 by tetrad analysis. Chemicals. Glucose, sucrose, potassium acetate and mercaptoethanol were of p.A. grade from Merck (Darmstadt, GFR). Agar, yeast nitrogen base without amino acids, peptone and yeast extract were from Difco (Detroit, USA). Amino acids, adenine, uracil, cycloheximide, eanavanine and calf thymus DNA were from Sigma (St. Louis, USA). Glusulase was from ENDO Laboratories (Garden City, NY, USA). Percoll was from Pharmacia (Uppsala, Sweden). Dlamidinophenyl indole was a gift from Dr. O. Dann (Erlangen, GFR). All other chemicals were of reagent grade. Media; Growth, Sporulation and Germination Conditions. The yeast strains were kept on YPD agar slants (containing 2% agar 2% glucose, 2% peptone and 1% yeast extract) at 4 °C. Standard sporulation experiments were performed as follows: Diploid strains were grown overnight at 30 °C in well aerated liquid medium containing 0.5% glucose and 1% yeast extract. The stationary cultures were diluted 1 : 1 with fresh medium containing 0.3% glucose and 1% yeast extract and grown for an additional 2 h. During this period glucose was used up completely and the cell densities nearly doubled. The ceils were now harvested and washed three times, and then resuspended in 1% potassium acetate at a density of 5 x 107 cells per ml. After 48 h sporulation was complete under these conditions. The experiments were performed either in Erlenmeyer flasks in rotary shakers (up to 1 1 of total volume) or in a New Brunswick type Microferm fermenter (10 1 of total volume). For the germination experiments, the spores were suspended in vegetative growth medium (1% glucose, 0.3% peptone, 0.8% yeast extract) containing 0.1% Triton X-100 and shaken at 200 rev/min at 30 °C.
mitotic recombinant in ade-2 isolated by F. Fel~l described in (Hartig and Breitenbach 1980) described in (Conde and Fink 1976) mitotic recombinant in ade-2 isomitochondrial with V-17 isomitochondrial with AP-3
For the glucose repression and derepression experiments the following media were used: 10% glucose, 1% yeast extract (YD) or 3% glycerol, 1% yeast extract (YG), respectively. For the purpose of tetrad analysis small scale sporulation experiments were performed by growing the diploid strains on YPD agar and replica plating on solid sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose and 2% agar). YPG (3% glycerol, 2% peptone, 1% yeast extract, 2% agar) was used for the detection for respiratory deficient colonies after mutagenesis. SD (0.67% yeast nitrogen base without amino acids, 2% glucose, 2% agar) was used for the isolation of diploid single colonies after crosses. SD+ leucine (30 ppm) was used for the isolation of cytoductants after crosses with strain JC8-AA1. Selection medium was SD+ 20 ppm adenine, 20 ppm uracil, 20 ppm histidine, 30 ppm lysine, 30 ppm tyrosine, 20 ppm cycloheximide and 80 ppm canavanine sulfate. This medium served for the discrimination of diploids derived from strain AP-3 (sensitive to cycloheximide plus canavanine) from haploids resistant to cycloheximide plus canavanine. All media were used with or without the addition of 2% agar.
Cell densities and percentage of asci were counted in a modified Neubauer hemocytometer. Mutagenesis and Mutant Selection. Strain AP-3 was grown to a ceil density of I x 10 "/ cells/ml in 10% liquid YPD (10% glucose, 2% peptone, 1% yeast extract). At this stage MnC12 solution was added to a final concentration of 1 mM and the culture kept at 30 °C without shaking for one day. Under these conditions stxain AP-3 grew to about 1 x 10 s cells/ml- The culture was diluted and plated onto 100 YPD plates (approximately 200 colonies per plate). After two days colonies showed up and were replica plated on YPG and sporulation agar. After another two days the sporulation plates were replica plated onto the selection medium. Two days later the replicas of wild type colonies exhibited growth on the selective medium, indicating the presence of living haploids (Fig. 1). Colonies growing on YPG, but not on selective medium were picked, subcloned four times and further analyzed genetically. The expected phenotype of these clones was respiratory sufficient and sporulation or germination deficient.
A. Hartig et al.: Germination-specific Mitochondrial Mutants
~
31 All recordings of cytochrome spectra were performed with strains not carrying the homozygous ade-2 markers. These were either: mitotic recombinants of the two heteroallelic ade-2 markers or in one case strain V-320. This was necessary, because the red pigment produced by ade-2 strains obscures the cytochrome spectra.
PD
YPG ~ o o medium Genetic and Physical Mapping Procedures. The techniques used have been described elsewhere (Schroeder and Breitenbach 1981a).
@
setection medium
Results
Isolation o f the Mutants Fig. 1. Mutant selection scheme. Full circles: growing colonies, open circles: nongrowing colonies. The arrow indicates a colony that is respiratory sufficient (growing on YPG), but differentiation deficient (nongrowing on selection medium)
Fluorescent Staining. The DNA-specific fluorescent stain diamidinophenyl indole was used to visualize nuclear and mitoehondrial DNA yeast ascospores. The method used was that of Williamson and Fennell (1975). Preparation o f Yeast Aseospores. Yeast cells were sporulated in 1% potassium acetate as described above, harvested and treated with mercaptoethanol and glusulase to remove ascus wails. The spore tetrads were then shaken gently with glass beads in 0.5% Triton X-100 to obtain single spores and layered on top of Percoil gradients (75-90% Percoll, 0.25 M saccharose). After centrifugation for 1 h at 10,000 rpm (Sorvall SS-34 rotor) a spore pellet had separated from the remaining vegetative cells, ascus wails and debris. Centrifugation was repeated once. The spores which were better than 99.9% pure were kept in 0.5% Triton X-100 at 4 °C. Wild type spores remained completely viable for at least a year. Details of the procedure will be published elsewhere. Light Microscopy. Germinating yeast spores were photographed using a Reichert Diavar phase contrast microscope equipped with an automatic camera. Measurement o f the respiratory activity o f the cells and micromanipulation were performed as described earlier (Hartig and Breitenbach 1980). DNA determination was performed as described by Schroeder and Breitenbach (1981b). Recording Cytoehrome Spectra. a) Vegetative cells were grown overnight in YG to a cell density of 5 x 107 cells/ml, diluted with fresh YG to a cell density of 1 x 107 cells/ml, grown for another 8 h and harvested at a cell density of 3 to 4 x 107 cells/ml (derepressed cells). Alternatively, the overnight cultures were diluted with YD (10% glucose) to a cell density of 2 x 107 cells/ml, grown for another 8 h and harvested at a ceil density of 4 x 107 cells/ml. The cells were treated with an excess of Na2S204 in suspension, quickly centrifuged, the pellet applied to the window of a home-made cuvette and frozen in liquid nitrogen. Spectra were recorded against several layers of Parafilm in a Beckmann LS 24 recording spectrophotometer, b) Spores were isolated on Percoll gradients as described above and treated in the same way as vegetative cells for the recording of the cytochrome spectra.
Out of 20,000 primary clones, we isolated 22 colonies that grew normally on glycerol b u t failed to produce living hyploids. Four of them were sporulation deficient (spo-). All four had gained the ability to mate with appropriate tester strains. We assumed that these were mutations in the mating type locus (or epistatic over the mating type locus) and excluded them from further analysis. The remaining 18 mutants produced asci in high yield b u t b y mass spore analysis on selective medium (Fig. 1) no germination was observed. Upon subcloning and analysis of individual spores four of the mutants (V-13, 17, 19, 22) showed a stable phenotype of germination deficiency, i.e. the spores did not form colonies on YPDagar.
Genetic Analysis For the purpose of genetic analysis it was necessary to isolate haploid clones carrying the mutation. Two of the four mutants mentioned (V-17 and V-19) were not totally germination deficient b u t produced living haploids at a low frequency (1 in 107 to 1 in 106) when spores were plated on selective medium. Some of the haploids were tested for mutant or wild type phenotype b y crossing them with rho ° - mating partners, isolating and sporulating the diploid clones and testing spore tetrads for their ability to form colonies on YPD-agar. The spores again showed the stable phenotype of germination deficiency. Some, however, were wild type with respect to germination. The theoretical implications of this behaviour will be part of the discussion. Haploid V-17/5 was used for further genetic analysis. It was crossed with strain JC8-AA1 (rho °) and haploids were isolated from the cross that carried the nuclear genome of JC8-AA1, b u t the mitochondrial genome of V-17/5 (Conde and Fink 1976). One such strain was crossed with strain F-101-1; the spores of the resulting diploid (V-319) again were germination deficient. This cytoduction experiment with strain JC8-AA1 revealed
32
A. Hartig et al.: Germination-specificMitochondrialMutants
•106c eLtS/m l
n moles Oz/rninJO6ceL[ s
,106cetl.S/m L 100 40
Fig. 2a. Growth on YPD (1%glucose) medium. Circles: wild type AP-3, triangles: mutant V-17, b Growth on YPG (3%glycerol)medium, c Respiration during growth on YPG (3% glyc-
50 20
12
24 h
12
b
24
48
h
c
c
n mol.es 02/min,106c ett s
°/oasci
30 100Olo
] \
/
• 20
12
A
h
48
erol)
Phenotypie Characterization of the Mutants
550 600 wavelength [nm]
600
Fig. 3AID. Low temperature cytochrome spectra of vegetative cells. A Wild type AP-3 rec. on YG (3% glycerol). B Mutant V-17 rec. on YG (3% glycerol. C Wild type AP-3 rec. on YD (10% glucose). D Mutant V-17 rec. on YD (10% glucose)
increase
24
that the germination deficiency of strain V-17 was inherited by a cytoplasmic genetic element. By a series of mitochondrial crosses a single genetic factor on mitochondrial DNA could be shown to be responsible for the germination deficieny. Its location is between the genes cob and oli2 (Breitenbach et al. in preparation).
E-l,0
550
12
~mo/.es 02/, . .^6
24
36 h
.
60 100% DNA e increase
~
40
20
12
24
36 h
Fig. 4. A Wild type AP-3 during sporulation. Triangles: Respiration, Circles: premeiotic DNA synthesis, Squares: percent asci, B Mutant V-17 during sporulation. Symbols as in Fig. 4. A
a) Vegetative Growth. As shown by the data presented in Fig. 2a and 2b growth of mutant and wild type on media containing 1% glucose or 3% glycerol as a carbon source was similar. Oxygen uptake of the cells during growth on glycerol medium was also nearly identical in mutant and wild type (Fig. 2c). The cytochrome spectra of mutant and wild type after growth on 10% glucose and 3% glycerol, respectively, were drastically different (Fig. 3). The wild type showed the well known pattern of glucose repression: On ] 0% glucose the characteristic absorption bands of cytochromes aa3, b, c 1 and c are 5 to 10 times less intense than on 3% glycerol. The mutant spectrum on 10% glucose was nearly as intense as the wild type spectrum on 3% glycerol, i.e. the mutant lacked carbon catabolite repression of cytochromes. On the other hand, the mutant spectrum on glycerol was nearly normal. The possible correlation of the lack of carbon catabolite repression with the germination deficiency will be discussed below. b) Sporulation. Our first idea was to look for biochemical defects during the sporulation phase, reasoning that the absence of some of the substances normally present in spores could cause the germination deficiency. However, most of the biochemical and cytological criteria of sporulation that we tested in the mutant appeared to be quite normal: Respiration, DNA synthesis and the percentage of mature asci during sporulation experiments reveal no significant differences between mutant and wild type (Fig. 4a, b). The morphology of the mutant spores as
A. Hartig et aL: Germination-specificMitochondrial Mutants
33 of an ascus. In some cases "blobs" representing mitochondrial DNA (Wiltiamson and Fennell 1975) could be seen in the asci, inside as well as outside the spores. Also in this respect we could not find any difference between wild type and mutant. The mutant spores seemed to contain mitochondrial DNA. The only difference between mutant and wild type spores which we have found so far is their behavior on Percoll density gradients. In the stepgradients used (75-90% Percoll) the wild type spores sedimented to the bottom of the centrifuge tube. Under the same conditions the mutant spores banded at the boundary between 85% and 90% PercoU. This was true for spores of both the strains V-17 and the isomitochondrial but nuclearly unrelated V-319.
Fig. 5 a and b. Mature asci stained with diamidinophenylindole and viewed in the fluorescence microscope, a Wild type AP-3. b Mutant V-17 judged by phase contrast microscopy also seemed to be quite normal. Figures 5a and 5b show mature asci of wild type and mutant which have been stained with diamidinophenyl indole. In those asci which were oriented properly four spore nuclei with about equal fluorescence could be seen. This means that nuclear DNA probably segregates normally to the four spores
c) Germination. As observed by phase contrast microscopy, wild type spores during germination on YPD (1% glucose) behaved as described in the literature (Rousseau 1972). The spores went through the following stages: a) "Germination proper", i.e. phase darkness and some swelling of the spores (30 rain to 1 h after suspension in YPD), b) "outgrowth", i.e. elongation of the spores (1-3 h in YPD), c) "budding". The first buds appeared around 4 h after the start of the experiment. Electron micrographs showed that the first two or three budding cycles differ from purely vegetative cycles in that the cell walls were considerably thicker than normal vegetative cells walls (data not shown here). This means that germination specific processes probably occur for upt to 10 h in germination medium. The formation of zygotes appears only after the first b udding. Mutant strain V-17 went through the stages of phase darkness and elongation of the spores (however at a slower rate than the wild type) but was arrested during the first budding cycle. Some of the spores did not bud; however, they were enlarged to a size greater than normal vegetative cells. At 6.5 h many spores had already lysed.
ng ONA/106 spores
n m°[ ee 02/rnin, 106spores
6C
,/
/
.9
40
.7
J
.5
2C
.3
.2 .1 a
.1 2
4
6
8
10
12
26 h
b
2
4
6
8
10 h
C
2
4
6
8
1~3 h
Fig. 6. a Growth of spores under conditions of germination. Optical density was measured at 546 nm. Triangles: wild type, circles: mutant V-17. b DNA synthesis under conditions of germination. Triangles: wild type AP-3, circles: mutant V-17, e Respiration of spores under conditions of germination. Triangles: wild type AP-3, circles: mutant V-17
34
A. Hartig et al.: Germination-specific Mitochondrial Mutants
Fig. 7. a Low temperature cytochrome spectra of spores of the wild type strain V-320 during germination. The numbers next to the spectra indicate hours in germination medium, b Low temperature cytoehrome spectra of spores of the mutant strain V-17 rec. during germination. Symbols as in Fig. 7a 500
600
nm
560
600
nm
The germination deficient spores could not be rescued by mating them with strains of a or ~ mating type. This was checked by micromanipulating mutant spores and placing one or two vegetative haploid cells of the same mating type next to every member of a tetrad. In no case did the growing colonies contain diploid cells. As can be seen in Fig. 6b, the wild type grows with a doubling time of about 2 h from the time of the first budding, whereas the mutant stops growing around 8 h By this time its cells number and biomass have less than doubled. DNA synthesis in germinating mutant spores also reflects this situation. Figure 6a shows that the mutant spores approximately doubled their DNA content during the first 6 - 8 h of the germination experiment, then stopped synthesizing DNA. Respiration of germinating spores (Fig. 6c) started during the outgrowth of wild type spores. The mutant spores reached only a low level of respiration, corresponding approximately to one tenth of the maximal respiration attainable by strain AP-3. The time course of the synthesis of spectroscopically visible cytochromes during the germination of wild type spores is shown in Fig. 7a. Mature spores contain a very small amount of cytochromes. During germination cytochromes c, b, and aa 3 are synthesized at different times. Cytochrome c appears after 4 - 6 h, b after 6 - 8 h and aa 3 only after 10-12 h. Figure 7b shows that the mutant spores do synthesize cytochrome c at the normal rate, but they do not synthesize cytochromes b and aa 3 at all over a time course of 24 h. In other words, mutation V-17 has a pleiotropic effect on the production of both cytochromes b and aa3.
Discussion The strain used for the mutant selection procedure as described here, is a normal diploid a/c~ strain. This means that only dominant nuclear mutations or cytoplasmic mutations can be detected. The procedure does not discriminate between sporulation deficient and germination deficient mutants. As has been described in this paper, we have predominantly isolated mutants of a single phenotype, namely respiratory competent mutants which are germination deficient and as a pleiotropic effect are deficient in carbon catabolite repression of cytochromes during vegetative growth. One of the mutants has been mapped on the mitochondrial genome. Nuclear mutations have also been isolated (Baranowska et al. 1977), but were not investigated for the purpose of this paper. Based on the results of our previous work on the role of the mitochondrial genome in yeast sporulation (Hartig and Breitenbach 1980), we also expected to find mitochondrial mutations which are respiratory competent but sporulation deficient. Such mutants have not been found up to date. Genetic characterization of the germination deficient mutants was possible due to the fact that with a frequency of about 10 .7 spores were found to grow out. Their progeny was composed of a majority of mutant cells still carrying the ger- mutation, and a minority of get + cells, which probably are revertants. We explain their occurrence as follows: In one cell, and even in one sPore a multiplicity of mitochondrial genome exists. Careful morphometric work (Brewer and Fangman 1980) showed that spores contain probably 1 0 - 1 4 mito-
A. Hartig et al.: Germination-specificMitochondrialMutants chondrial genomes. In those strains that revert at a low frequency, the presence of only one or two wild type mitochondrial genomes may suffice to enable the spore to outgrow normally. During growth of the resulting haploid colony the ger+ and ger- mitochondrial genomes segregated to different cells lines. The method was only applicable for one of the mutant strains. The differentiation of mitochondria during germination of yeast spores is one of the most striking results of the present work. The cytochrome spectra (Figs. 7a, b) and the complete absence of respiration in resting spores indicate that the mitochondria of the spores are in an extremely repressed state. Electron microscopic evidence further corroborates this fact. Sando et al. (1980) found that the mitochondria of spores contain little internal structure (cristae), in fact they have been termed "promitochondria-like" in the older literature (Hashimoto et al. 1958). Brewer and Fangman (1980) have shown by serial sectioning 4 different asci, that the spores in most cases contain one large mitochondrion that is described as "fermentation4ike". During germination the ultrastructural features of the mitochondria slowly change to "normal" (Kreger-Van Rij 1978). Our results show that the three cytochromes c, b, and aa3 are synthesized at different rates. The nuclear encoded cytochrome c appears during the "outgrowth" phase, but the (partly) mitochondrially encoded cytochromes b and aa 3 appear at six and after 10 h in germination medium, respectively. These results are in excellent agreement with the work of Brambl and Josephson (1977) and Brambl (1977), wo have measured cytochrome spectra during the germination of Botryodiplodia theobromae spores. They found virtually the same kinetics for the appearance of cytochromes c, b and aa 3 in those spores. This could mean that the extreme repression of mitochondria and the slow and asynchronous reappearance of mitochondrial cytochromes during germination is a gerneral phenomenon in fungal spores. In the case of Saccharomyces cerevisiae spores, we do not know at present whether de novo synthesis of cytochromes or some other process (modification, assembly in the inner mitochondrial membrane) is responsible for the appearance of the visible cytochromes. In the case of Botryodiplodia spores it has been shown that the novo mitochondrial protein synthesis is indeed necessary for the reappearance of cytochromes b and aa3. The germination deficient mutant described here is blocked at the time of the first budding cycle, which is a rather late stage of germination. It produces nearly normal amounts of cytochrome c, but no cytochromes b and aa 3 during germination. This is in good agreement with the fact that the partly mitochondrially synthesized cytochromes b and aa3 also appear later in germination and that fermentatively usable carbon sources are absolutely needed for the first few hours of germination
35 (Banerjee 1971, and our own unpublished results). One hypothesis which would account for this fact, is that the mutant is unable to reactivate the mitochondrial protein synthesizing system during germination, but this hypothesis has to be tested experimentally. The late manifestation in germination of mutant V-17 points to the fact that germination-specific processes occur for up 10-12 h after suspension of the spores in germination medium. Similar conclusions have been reached in the literature (Rousseau and Halvorson 1973 a and b; Rousseau 1972; Savarese 1974; Kreger van Rij 1978) based mainly on electron microscopic observations: Three stages ("germination proper", "outgrowth", and "budding") can be distinguished. The outer spore wall, which is very osmiophilic, ruptures during "outgrowth", but the new wall synthesized during the first two to three budding cycles is similar to the inner wall of spores and only gradually changes to a vegetative cell wall. What is the role of the yeast mitochondrial genome in germination and how does mutation V-17 interfere? Concerning spomlation there is now evidence that specific mitochondriai genes and mitochondrial protein synthesis are required for the normal completion of the process (Puglisi and Zennaro 1971; Newlon and Hall 1978; Hartig and Breitenbach 1980). Sporulation-and germination-specific products of mitochondrial transcription and translation have been found (Schroeder and Breitenbach 1981a). In contradiction to that, Tingle et al. (1974) reported that spores which are devoid of mitochondrial DNA can grow out and form haploid colonies. However, some objections have been raised against their conclusions (Newlon and Hall 1978; Hartig and Breitenbach 1980). We believe that these experiments do not rigorously exclude the presence and expression of mitochondrial genes during sporulation and germination. Evidence favoring a specific participation of the mitochondrial genome in germination has been scare up to now. Rousseau and Halvorson (1973a) found that erythromycin is an inhibitor (to some extent) of the germination o f yeast spores, indicating that mitochondrial protein synthesis may be necessary for germination. The results described in the present paper point to a specific role of a mitochondrial gene in germination. A mutant in that gene, which blocks germination during the formation of the first bud, leaves vegetative growth nearly unaffected. Genetic and physical mapping locates the mutational site to within 2,000-3,000 bp in the region between the genes cob and oli2 on the mitochondrial genome (Breitenbach et al. in preparation). No sequences and no other genetic data are available at present from this region. Therefore, it is not possible to draw any conclusion as to the expression of this gene. It may be transcribed and translated, only transcribed, or it may be a nontranscribed regulatory DNA sequence. The pleiotrophic, "regulatory" character of the mutation is documented by its
36 phenotype: during vegetative growth, the glucose repression of cytochromes is missing, the spores exhibit a density different from wild type o n Percoll gradients, and during germination appearance of cytochromes b and aa 3 is blocked.. We believe that it is the repressed state of spore mitochondria that necessitates a specific mitochondrial gene for the reactivation of the mitochondrial genetic system during germination and that m u t a t i o n V-17 confers a defect in such a regulatory gene.
Acknowledgement: The authors wish to express their gratitude to Dr. R. J. Schweyen and Dr. H. Lehrach for their help and encouragement during the course of this work and for many fruitful discussions, to Dr. W. Schnedl for his help with the fluorescent staining of DNA and to Prof. O. Hoffmann-Ostenhof for critically reading the manuscript. This work was supported by the Ludwig Boltzmann-Gesellschaft and by a grant from the Jubiliiumsfonds der ~sterreichischen Nationalbank, No. 1106.
References Banerjee M (1971) Ph.D. thesis, McMaster University, Hamilton, Ontario Baranowska H, Ejchart A, Putrament A (1977) Mutat Res 42: 343-348 Brambl (1977) Arch Biochem Biophys 182:273-281 Brambl R, Josephson M (1977) J Bacteriol 129:291-297 Brewer BJ, Fangman WL (1980) Proc Natl Acad Sci USA 77: 5380-5384 Conde J, Fink GR (1976) Proc Natl Acad Sci USA 73:36513655 Diala ES, Wilkie D (1977) In: Bandlow W, Schweyen RJ, Wolf K,
A. Hartig et al.: Germination-specific Mitochondrial Mutants Kaudewitz F (eds) Mitochondria. De Gruyter, Berlin New York, pp 563-570 Hartig A, Breitenbach M (1980) Curt Genet 1:97-102. Hashimoto T, Conti SF, Naylor HB (1958)J Baeterio176:406416 Kreger-Van Rij NJW ( 1978) Arch Microbio1117: 73 -77 Newlon MC, Hall BD (1978) Gen Genet 165:113-114 Pollak JK, Sutton R (1980) Trends in Biochem Sci 5:23-27 Puglisi PP, Zennaro E (1971) Experientia 27:963-964 Putrament A, Baranowska H, Prazmo W (1973) Mol Gen Genet 126:357-366 Rousseau P (1972) J Bacteriol 1.09:1232-1238 Rousseau P, Halvorson HO (1973a) Can J Microbiol 19:13111318 Rousseau P, Halvorson HO (1973b) J Baeteriol 113:12891295 Rousseau P, Halvorson HO (1973c) Can J Microbiol 19:547555 Sando N, Oguchi T, Nagano M, Osumi M (1980) J Gen Appl Microbiol 26:403-412 Savarese JJ (1974) Can J Microbiol 20:1517-1522 Schroeder R, Breitenbach M (1981a) In: Levinson HS, Sonenshein AL, Tipper DJ (eds) Sporulation and Germination (in press) Proceedings of the 8th Int. Spores Conference. A.S.M. Publications Schroeder R, Breitenbach M (1981b) J Bacteriol 146:775-783 Tingle MA, Kiienzi MT, Halvorson HO (1974) J Bacteriol 117: 89-93 Williamson DH, Fennell DJ (1975) In: Prescott DM (ed)Methods in cell biology, Vol. XII Academic Press, New York,pp. 335352
Communicated b y R.J. Schweyen Received May 15/July 20, 1981
