Regulation of mitochondrial biogenesis: Yeast mutants deficient in synthesis of δ-aminolevulinic acid

J. Mol. BziL (1973) 80, 1739

Regulation of Mitochondrial Biogenesis : Yeast Mutants Deficient in Synthesis of kbninolevulinic

Acid

H. K. SANDERS,P. A. &ED, M. Bmmm-f, J. HERNANDEZ-RODRIGUEZ$, R. F. GOTTAL AND J. R. MATTOON Department of Phy&ologicd Chemistry The Johns Hopkins University School of Medicine Ba&nore, Md, U.S.A. (Received 27 February

1973, and in revised form 25 June 1973)

A new class of Saccharomyces cerevi&e mutants deficient in biosynthesis of ah cytochromes was isolated from cultures grown in medium containing ethidium bromide. Cytochrome c synthesis may be restored to normal by growing mutant cells in medium supplemented with S-ammolevulinic acid. Cytochrome deficiency results from mutation in two genetic determinants, one nuclear, the other mitochondrial. When cells possess normal (p’) mitochondrial DNA, expression of the abnormal nuclear determinant (cyd-1) is largely masked, so that cells can grow on glycerol as primary carbon source and all cytochromes are present. Nevertheless, the presence of the cyd-1 mutation may be detected in p+ strains, since synthesis of all cytochromes is enhanced to some extent by &sminolevulinic acid. Destruction of mitochondrial DNA unmasks the underlying defect so that cyd-1 p- strains are almost completely lacking in detectable cytochromes. Although spectra of cyd-1 p+ strains resemble those of cytochrome c (cyc) mutants, cyd-1 mutants represent a new complement&ion group different from six known cyc groups. Cytochrome c biosynthesis in only one of these six types of cytochrome c mutants, cyc4-1, was restored to normal by I-aminolevulinic acid. Therefore, since cyc4-1 and cyd-I are complementary, and segregate independently, 6aminolevulinic acid synthesis appears to be controlled by at least two nuclear genes, and by one or more genes located in mitochondrial DNA. Glycine does not replace &eminolevulinic acid in stimulating cytochrome biosynthesis in cyd-1 or cyc-4 mutants. A regulatory system involving exchange of information between mitochondria and the nuclear-cytosolic compartment is indicated by the results. Studies with isolated mitochondria indicate that a limitation of intracellular %-aminolevulinic acid supply is reflected in mitochondrial composition, not just in numbers of organelles.

1. Introduction The regulation of mitochondriel biogenesis requires the co-ordinated control of the production of several diverse types of components: proteins, nucleic acids, lipids and a variety of cofactors. Because all of the respiratory cytochromes contain ironporphyrin prosthetic groups, the control of porphyrin biosynthesis is of centra importance in determining the development of the mitochonclrial inner membrane. t Present Cstholique $ Present de MCxico, 2

address : Labor&ok d’Enzymologie, Facult6 des Soienoes Agronomiques, Universiti de Louvein, Heverlee, Belgium. address : Departamento de Bioqufmice, Faaultad de Medicina, Universidad National MBxico, D, F, 17

18

H.

K. SANDERS

ET

AL.

Since the first committing step in porphyrin biosynthesis is the formation of Saminolevulinic acid, it is probable that this reaction represents a primary point of control not only of porphyrin synthesis, but of the entire respiratory chain. The present work describes mutants of the yeast, Saccharomyces cerevisiae, that are apparently defective in the formation of Alvt. Synthesis of all mitochondrial cytochromes is somewhat defective in these mutants, but normal cytochrome production may be attained by growing mutant cells in medium supplemented with Alv. A fundamental feature of mitochondrial biogenesis is the intracellular compartmentation of different component reactions. Although mitochondria contain a unique species of DNA (Borst, 1972) which codes for key components of an autonomous, mitochondrial protein synthesis system, namely ribosomal RNA (Wintersberger, 1967) and tRNAs (Dawid, 1972), the amount of genetic information in this DNA is limited by its small size (Borst, 1972). Consequently, mitochondrial biogenesis is highly dependent upon nuclear genetic information (Beck et al., 1971), i.e. the great majority of the various mitochondrial proteins are coded by nuclear genes. Moreover, the bulk of mitochondrial protein arises by translation on extramitochondrial, cytosolic ribosomes (Borst, 1972).

Uro, copto, and *prolo-porphyrinogens

Parphobilinogen

I

MITOCHONDRION

(oxidasel

Alv

I Protoparphyrin lX (Fe) Succinyl CaA

Cyto:hrome

o A

A-r

Cytochrame b,cl

Nucleor genes

1 -~

Cytosol ribosomes

FIQ. 1. Compartment&ion of heme and cytochrome into cytochromes has not been firmly established. shown) may contribute to cytochromes a, b and cl.

7

Cytochrame c _I

Protein;

biosynthesis. The site of heme incorporation Intramitochondrial protein synthesis (not

Porphyrin synthesis, like mitochondrial protein synthesis, also involves an intracellular division of labor, as shown in Figure 1. Both the initial and terminal reactions are intramitochondrial, while intermediate biosynthetic steps are extramitochondrial. Alv biosynthesis appears to occur largely within mitochondria (McKay et al., 1969; Sano & Granick, 1961), although evidence has been presented that a fraction of cellular Alv synthetase activity is localized in the cytosol (Hayashi et al., 1969; t Abbreviation

used : Alv, S-aminolevulinic

acid,

S-AMINOLEVULINATE-DEFICIENT

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19

Scholnick et al., 1969).The enzymes catalyzing conversion of Alv to protoporphyrinogen appear to be extramitochondrial (Sano & Granick, 1961), but the final step, conversion of protoporphyrinogen to protoporphyrin, is catalyzed by an intramitochondrial oxidase (Sano t Granick, 1961). It should be added that the terminal step in protoheme synthesis, the insertion of iron catalyzed by ferrochelatase, also occurs within the mitochondria (McKay et al., 1969; Labbe, 1971). It is obvious that the separation of various steps in mitochondrial biogenesis into different physical compartments imposes a need for a system of communioation between them. It is likely that this communication involves the flow of both low molecular weight metabolites and marcromolecules, and it may involve the movement not only of structural components but of regulatory molecules as well. The present study of mutants showing Alv-dependent cytochrome biosynthesis provides a striking example of the interdependence of two genetic compartments. Destruction of mitochondrial genetic information by treatment with ethidium bromide enhances dramatically the expression of a nuclear gene controlling the biosynthesis of all cytochromes. This result indicates the existence of a system of communication between mitochondrial DNA and the nucleus, whose operation is of fundamental importance in regulating mitochondrial biogenesis.

2. Materials and Methods (a) Mutant strains and genetic methods Cytochrome-deficient mutants were prepared from two haploid S. cerewisiae strains, Ql and Q2, selected for efficient growth on glycerol. Both strains are of CL mating type and are auxotrophic for adenine (ode-l). Strains Ql and Q2 had been derived from a normal diploid, strain JP292, heterozygous for a&e-l, his-l, Zys-2, trp-1 and trp-2 (Beck et al., 1971). As indicated in Results, a substrain of Q2, Q2M1, was used in several experiments. This substrain, although it respired, was characterized by relatively inefficient growth and cytochsome biosynthesis. Haploid strain D311-3A (a h&s-l lye-2 trp-2) was used as a tester strain in preparing heterozygous diploids from cytochrome-deficient mutants. Cytochrome c-deficient mutants, each containing one of 6 different cyc mutations (formerly designated cy) were kindly provided by Dr Fred Sherman of the University of Rochester, and by Dr John Parker of Miles Laboratories. A list of these strains is given in Table 7. Before use, each strain was purified by plating, followed by isolation of a clone from a single colony. In the case of the cyc4-I strain, two subclones were isolated from a large colony and a somewhat smaller colony. These subclones are designated, respectively, B271 (L) and B271 (S). The method of Nagley & Linnane (1970), used for isolating neutral petites lacking detectable mitochondrial DNA, was used. This method involves growth of normal yeast for 18 h in yeast-peptone-dextrose medium (see below) containing ethidium bromide. The concentration of ethidium bromide used was either 20 pgjml or 60 pg/ml, as indicated. Standard methods of mating, sporulation, ascus dissection, and tetrad analysis were used (Hawthorne & Mortimer, 1960; Mortimer & Hawthorne, 1969). A Pepper Inoculator (Pentex, Inc., Kankakee, Ill.) was used for replica plating. (b) Cytochrome determinations Cytochrome content of mutant and normal strains was estimated visually after freezing cell pastes in liquid nitrogen. A Zeiss spectroscope was used, as described previously (Parker BEMattoon, 1969; Sherman, 1964). An arbitrary scale of relative concentration, based on density of absorption bands, was used as shown in the legend to Table 7. To obtain a quantitative record of the cytochrome content of p+ cells, difference spectra were obtained using a split-beam spectrophotometer (Beck et al., 1971). Oxygen was used as oxidant, and reduction was accomplished by adding glucose to the sample cuvette and

20

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ET AL.

SANDERS

bubbling the cell suspension with nitrogen. For p- and CYDcells absolute spectra were recorded using dilute whole milk placed in the reference cuvette se a turbidity blank (P. Rickard & J. Mattoon, unpublished method). In some experiments dithionite was used ss a reductant. (c) Media and growth conditions These were essentially as described previously (Parker & Mattoon, 1969). In most experiments cultures were grown on one of the following media : dextrose medium, consisting of 1% yeast extract (Difco), 2% peptone (Difoo) and 3% (w/v) dextrose; glycerol medium similar to dextrose medium except that dextrose was replaced by 3% (v/v) glycerol; minimal medium O*67o/o yeast nitrogen base without amino acids (Difco), 2% dextrose. When strains auxotrophic for adenine were used, dextrose medium and glycerol medium were supplemented with O-08 or O.lOo/o adenine sulfate. For genetic studies the minimal medium was supplemented with adenine sulfate and various amino acids, as previously described (Parker & Mattoon, 1969). In several experiments dextrose medium was supplemented with filter-sterilized S-aminolevulinic acid, at a concentration of O-5 mrvr or as indicated in the legends to Figures and Tables. Cultures were grown at 30°C. Liquid cultures were grown on a rotary shaker (Beck et al., 1971) or in a New Brunswick Microferm fermentor (Mattoon & Sherman, 1966). Dry weight was determined by collecting cells on Millipore filters, and drying at 80°C for at least 24 h. (d) Preparation Yeast mitochondria method of Balcavage 1971).

of yea& mitochondria

were prepared from cells grown in a fermentor et o.!. (1970), and purified on Renogralln gradients (e) Chemicd

according to the (Beck & Mattoon,

reagenta

Renogrti (free acid) was the generous gift of E. R. Squibb & Sons, Inc., and was converted to a salt using N-methylglucamine (Baker). &Aminolevulinic acid wss obtained ae the hydrochloride salt from Sigma and ethidium bromide from Calbiochem.

3. Results (a) Diecovey of mutants de&Sent in all cytochromea In the course of an experiment designed to produce cytoplasmic petite mutants completely lacking mitochondrial DNA (Nagley BELirmane, 1970) we encountered some unusual new mutants. As shown in Table 1, certain strains, after growth in dextrose liquid medium containing ethidium bromide and subsequent plating and TABLE I

Growth and cytochrome content of ethidium bromide-treated cells Parent

strain no.

&1

62 D311-3A

Ethidium bromide conon bdml) 20 60 20 60 20 60

Colony size

Medium Small Small Medium Medium Medium

Cytochrome

c

+ 0 0 z +

Cultures were grown at 30°C for 18 h on a rotary shaker in dextrose medium containing the indicated ooncentrations of ethidium bromide. Treated oells were then plated on dextrose medium. Cytoohrome c was estimated visually with a Zeiss spectroscope.

1-AMINOLEVULINATE-DEFICIENT

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21

growth on dextrose agar medium, developed two distinct colony types. In two cases ethidium bromide mutagenesis resulted in an almost uniform population of very small colonies which took four to five days to grow to a diameter of about 0.5 mm to 1.0 mm. At different ethidium bromide concentrations uniform populations of medium-sized (1.0 mm to 20 mm) colonies were formed. Strain D311-3A produced medium-sized colonies at either ethidium bromide concentration. Triplicate sets of individual clones of each colony type were isolated, grown on dextrose medium and examined spectroscopically for cytochromes. In clones isolated from medium-sized colonies, a typical p- absorption spectrum (Sherman & Slonimski, 1964) containing only a cytochrome c band was seen, but when the small-colony strains were observed in the spectroscope no bands could be detected. These data indicate that in two of the three strains, ethidium bromide, under certain conditions, caused the loss of all mitochondrial cytochromes including cytochrome c. Since both mutant types are respiration deficient (petite) they were tentatively designated p- (typical petite) and CYD- (cytochrome-deficient petite). (b) Irreversibility

of cytochrome deficiency

When clones of the CYD- strains were subcultured and subsequently re-plated on dextrose medium, an occasional colony appeared which was slightly larger than the general population. Subsequent subculturing of these exceptional forms, followed by spectral observation, revealed very faint bands absorbing at about 546 and 555 nm. These modified forms appear to be quite stable and apparently represent a less extreme form of the cytochrome deficiency found in the general population. Nevertheless, they are clearly distinguishable from the p- petites, which have strong cytochrome c absorption bands. The apparent stability of the CYD- cytochrome deficiency through several successive subcultures was tested further by growing a cytochrome-deficient strain derived from strain Ql in a fermentor through 10 to 12 mass doublings. When cells from the fermentor were examined with the spectroscope, no cytochrome bands were detected. Subsequent plating of a sample of these cells revealed that about 20% of the colonies were larger than the general population. However, when clones derived from representative larger colonies were subcultured and their spectra examined, two very faint bands absorbing at about 546 and 555 nm were again seen. Apparently, some deficient cells undergo a slight adaptation or secondary mutation to produce a form that grows more efficiently and produces traces of pigments, presumably heme proteins. Notwithstanding this small change, it is clear that a general cytochrome deficiency is characteristic of this strain, and its apparent stability indicates that it has a genetic basis. It was estimated that no less than 35 cell doublings had occurred between the initial mutant selection and the completion of growth in the fermentor. Since no deficient strain containing significant concentrations of cytochrome c has been observed, the mutation is phenotypically distinct from p-. Therefore, the symbol, CYD, will be used to describe the wild-type form of the presumed hereditary determinant, which controIs production of all mitochondrial cytochromes. A strain that contains CYD may contain all cytochromes (CYD p+), or only cytochrome c (CYD p-). Spectra of the three phenotypes are presented in Figure 2. Lack of CYD, the normal determinant, is indicated by CYD- in the Figure.

H.

22

640

620

600

K.

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ET

580

560

c+c1 AI,

540

520

0

Wovelength (nm)

FIU. 2. Whole-&l cytochrome spectra of normal 8nd two different petite forms of strain Ql. Celle were grown at 30°C for 48 h in dextrose liquid medium. Speotr8 were obtained at 16’C with a split-beam speotrophotometer oonstruoted by W. X. Belcevege. Cell suspensions of 2.6% (wet w/v) for p+ end 6% for p- end CYD- were made in 60 mrd-phosphate buffer, pH 4.6, and aqueous suspensions of whole milk were used as turbidity references. Ql p+ cells were reduoed with glucose, pet&s by endogenous substrates or dithionite. Strains Ql p+ and Ql p- contain the CYD determinant, as described in the text.

Since extensive ethidium bromide treatment during growth was used in producing CYD- strains, it is very likely that they lack mitochondrial DNA (Nagley & Linnane, 1970; Goldring et al., 1970) as well as the normal CYD determinant. At this point two questions may be posed: (1) is CYD a cytoplasmic determinant which is lost during ethidium bromide treatment? (2) Is loss of p factor essential to expression of the CYD- phenotype? The following genetic experiments were designed to answer these questions.

S-AMINOLEVULINATE-DEFICIENT

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23

(c) Recessivenessof cytochrome defiiency When cytochrome-deficient (CYD-) strains were crossed with normal (p’) strains, it was found that the resulting diploids were predominantly p+, as indicated by relative sizes of colonies formed by direct plating of zygotes (Table 2). Large-colony (L) isolates exhibited normal (p’) spectra. When ten selected small (S), or very small (VS) strains were subcultured and observed in the spectroscope, five were found to contain all cytochromes, while the other five had only cytochrome c, and were therefore p- , Presumably these occasional petites arose by spontaneous mutation of a p + diploid to p-, a commonly observed phenomenon. Therefore, CYD- is recessive and the CYD- petites have little or no suppressiveness (Sherman & Ephrussi, 1962). TABLE

2

Recea&vene.ssof cytochrome de$ciency determinant

cross D311-3A D311-3A

(p’) x QlCYD-s (p+) x Q2CYD-1

Distribution L

of colony S

99 239

0 3

size vs 2 2

Percentage s + vs 1.98 2.09

Zygotes formed by mass meting were plated directly on minimal medium and incubated for 6 deys st 30°C. Parental strains conteined complementary auxotrophic markers so thst only prototrophic diploids produced colonies. L, S snd VS design&e large, small, and very smzxll colonies, respeotively.

(d) Presence of the CYD determinuti in p- strains When haploid CYD- cells were mated with cells of a p- haploid derived from strain D311-3A, the resulting diploids exhibited a strong cytochrome c absorption band, typical of p-. This indicates that the p- tester contributed a normal CYD determinant to the zygote. No significant suppression of CYD replication in the diploid occurred, since subsequent plating of diploid cells revealed no CYD- daughter cells among 24 clones examined. CYD- is therefore recessive even in crosses with p -. When the p- strains, &I p- and Q2 p-, were crossed with a p+ tester, p- diploids appeared at a frequency of less than 2%, again indicating little or no suppressiveness. It is probable, then, that the p- strains lack mitochondrial DNA (Nagley & Linnane, 1970; Goldring et al., 1970). Consequently, it is unlikely that CYD resides in mitochondrial DNA. Crosses between two p- strains gave only p- diploids. (e) Non-segregation of cytochrome de$ciency from heterozygow di$oids If the cytochrome-deficient phenotype is determined by the nucleus, it should reappear among meiotic products formed by sporulating beterozygous diploids made by crossing CYD- and p+ haploids. Table 3 presents the results of analyses of tetrads derived from two different diploids prepared by mating independently-derived CYDstrains, QlCYD-, and &2CXD-,, with the same haploid tester, D311-3A p+. Since all segregants from 27 different tetrads utilized glycerol, and therefore contained a functional respiratory chain, no transmission of CYD- to any of the progeny occurred. Thus, the CYD- phenotype, like p-, does not segregate during meiosis (Ephrussi,

24

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TABLE 3 Tetrad analysis of &ploids produced by crossing normal and cytochrome-de$cient strains Diploid no. GT6 GT6 Totals

Cytochrome-deficient parent QlCYD-, Q2CYD-

No. of tetrads analyzed

No. of respirationdeficient segregants

13 14 27

1

0 0 0

Respiration deficiency was tested by replicating segregant clones on glycerol medium. In each tetrad, 4 independent auxotrophic marker genes (Mendelian) segregated normally (2: 2). The normal parent was strain D311-3A (p’) in each case.

1953). Although one might conclude from this result and from the apparent specific induction of CYD- by ethidium bromide, that the deficiency in all cytochromes is controlled solely by a cytoplasmic hereditary determinant, results described below indicate that the nucleus also has a key role. (f) Evidence for defective porphyrin

biosynthesis in CYD-

mutants

In an attempt to rationalize the apparent non-Mendelian (cytoplasmic) control of all cytochromes, the possibility was considered that some aspect of porphyrin or heme biosynthesis had been affected in CYD- mutants. A specific effect on cytochrome c biosynthesis seemed less likely, since both the structural gene for iso-l cytochrome c, and five other genes controlling cytochrome c concentration in yeast are nuclear (Sherman, 1964). In animal tissues three enzymes of the heme synthesis pathway have been localized mainly or entirely in mitochondria and are possibly subject to non-Mendelian control : &aminolevulinic acid synthetase, coproporphyrinogen oxidase and ferrochelatase (San0 & Granick, 1961; McKay et al., 1969). Since the spectrum of the CYD- yeast strain shown in Figure 2 exhibited no detectable absorption bands, there was no apparent accumulation of porphyrin or metalloporphyrins, as might be expected if the oxidase or ferrochelatase was defective. Therefore, A.lv was included in the growth medium to determine whether it could restore synthesis of cytochromes to CYD- cells. The data in Figure 3(a) show that addition of Alv induces the production of cytochrome c, but does not restore the a and 6 cytochromes. If sutlicient Alv is included in the growth medium, the cytochrome c concentration of the CYD- cells can be restored to the level found in typical pcells (CYD p-). Figure 3(b) shows that as Alv concentration is increased, cellular cytochrome c concentration can be restored to wild-type levels (Beck et al., 1971). However, the apparent efficiency of conversion of added Alv to the heme of cytochrome c never exceeds l%, and decreases with increasing Alv concentration. (g) Evidence for a masked &aminolevulinic

acid synthesis defect in p+ cells

Since Alv restores cytochrome c, but not the other cytoohromes, it is apparent that CYD- strains lack normal p factor. In addition, these strains have a second defect that appears to involve a genetic determinant controlling porphyrin production at the level of Alv biosynthesis. Since the original p+ strains, Ql and Q2, grew very well

5OOp1.4 -Alv

_ ~OOUM-Alv

I

I

I

I

I

I

630

610

590

570

550

530

510

Wavelength (nm) (a)

S- Am~nolevuhc

oc d imp)

(b)

FIQ. 3. Effect of Alv concentration on aytochrome synthesis in CYD- yeast. Cells were grown on a rotary shaker for 48 h in dextrose medium supplemented with Alv, as indicated. Cell suspensions, 6% (wet w/v) in (a), 6% or 10% in (b), were used. Spectra were measured 8g8inst a milk suspension aa described in the legend to Fig. 2. (a) Spectral traces of cells grown in dextrose medium containing different Alv conoentrations. (b) Cytochrome c concentrations of QlCDcells calculated from spectral traces,

28

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ET

on glycerol, there was no obvious reason to suspect that these strains contained any signScant defect in Alv biosynthesis or cytochrome before ethidium bromide treatment. However, when the p + form of strain Q2 w&s plated on dextrose medium, two populations of colonies differing in size could be detected. Representative colonies of the two types were selected and subcloned. When these subclones were subsequently grown in dextrose liquid medium they could be distinguished by their respective yields of cells, as indicated in Table 4. This result suggested a plausible 4

TABLE

Relative growth ejfiiency of two p+ fom

Cell dry weight (miidml)

Colony size and no.

Large Large Small Small

1 2 1 2

Cells were grown in liquid

of strain Q2

10.36 10.14 7.14 6.96 dextrose

medium

for 48 h at 3O’C.

explanation for the observation, illustrated in Table 1, that strain Q2 produced two classes of petite, p- and CYD- . Perhaps the “small” type contained a mutation affecting Alv production and gave rise to the CYD- petites upon ethidium bromide treatment. The large type, then, presumably represented normal p+ cells, which might have arisen by reversion of the small type, and upon ethidium bromide treatment produced p- petites. This hypothesis seems to be supported by the results TABLE

Effect of &aminolevulinic

acid on growth of large and small-type Q2 p+

p+ Strain &2 large Q2 small (1) Q2 small (2) Cells were grown on dextrose O-6 mm

5

Cell dry weight Control

(mg/ml) +Alv

9.30

9.00

2.23

9.16

6.93

9.26

medium for 48 h at 3O’C. When present the Alv concentration

was

shown in Table 6. Alv increased the yield of either of two small-type substrains up to that given by the large-type substrain, but Alv addition did not increase the yield of the latter. The fact that some p+ cells of strain Q2 also respond to Alv indicates that some defect in porphyrin synthesis existed in this strain before ethidium bromide treatment. However, the expression of this defect seems to be quite limited when cells

6-AMINOLEVULINATE-DEFICIENT

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are p+, since they grow quite adequately when glycerol is used as carbon source. Apparently, small-type strain Q2 has a genetic defect which predisposes the cells to becoming highly deficient in porphyrin synthesis upon ethidium bromide treatment. It appears, therefore, that destruction of p factor is required to obtain full expression of an underlying defect which is largely masked in the p + cells. This, then, constitutes an affirmative answer to the second question posed previously: less of p factor is essential to expression of the CYD- phenotype. (h) Mode of inheritance of the masked defect Since the expression of the defect in Alv synthesis is largely masked in strain Q2 (p+), growth on glycerol is not an appropriate test for its existence. In light of this new information, the tetrad analysis given in Table 3 must be reconsidered. While it is clear from this experiment that there are no CYD- segregants present among the meiotic progeny, the glycerol growth test would not distinguish normal segregants from segregants which might contain the masked defect. The data in Table 6 TABLE

6

Types of petites recoveredfrom t&-ads after ethidium bromide treatment Ethidium Segregant

bromide

(20 cLg/ml)

No. of clones

GTB-IA 1B IC 1D

3 3 3 3

GTG-IA 1B 1c 1D

4 3 4 3

Phenotype

P-

CGDCYD P-

&D2CYD-,lp+

concentration (50 x0-4 No. of Phenotype clones 3 3 3 3 3 3 2 3

P-

&DCYD ; P-

&DCYD -

Segregants (p+) were grown for 18 h in dextrose medium + ethidium bromide at the indioated concentrrttions, diluted snd plated on dextrose medium. Selected small clones were isolated, grown on dextrose medium plates and used for speotroscopic examination. Clones were scored as CYD if they lacked detectable cytochrome c or contained only traces of cytochrome. Clones scored as p- contained normal levels of cytochrome c. The CYD- parental strains for GTS and GT6 segregents were QlCYD-:, and QSCYD-,, respectively.

show that when selected tetrads are treated with ethidium bromide to inactivate or destroy p factor, two segregants become defective in synthesis of all cytochromes (CYD-), while the other two segregants produce typical petites (p-) which exhibit normal levels of cytochrome c. Therefore, the CYD- phenotype results from the interaction of two genetic determinants. One of these is non-Mendelian and is determined by mitochondrial DNA (p factor) ; the other is a Mendelian (nuclear) gene which controls Alv biosynthesis. Henceforth, then, the nuclear determinant, in its mutant and normal forms, will be designated respectively cyd and CYD. The CYDgenotype may now be more appropriately designated by cyd p-.

I

590

I

570

I 550

+ Alv

530

510 Wavelength

630

I

610

I

Large type 02

I 590

I 570

I 550

I 530

+ Alv

II 510

FIU. 4. Effects of Alv supplementation on small and large-type Q2 p+ substrains. Reduced minus oxidized difference spectra were obtained using 5% (wet w/v) cell suspensions. Glucose was used a.8 reductant. Cells were grown at 30°C on a rotary shaker in dextrose medium with or without 0.5 mM-Alv.

I

610

I

630

Small type 02

6-BMINOLEVULINATE-DEFICIENT

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29

(i) Variable expression of the cyd gene in p+ yeast The data in Table 5 indicate that the cyd gene may be detected even in a p + strain by measuring the increase in cell yield obtained when Alv is added to growth medium. The lack of an Alv response shown by the large-type substrain was initially thought to mean that the substrain contained the normal allele, CYD. As shown in Figure 4, comparison of the cytochrome spectra of whole cells of small-type Q2 (trace A) and large-type Q2 (trace C), grown in unsupplemented medium, indicates that all cytochromes are lower in the small-type cells than in the large-type cells. In particular, in the spectra of the small-type Q2 cells, the absorbance in the cytochrome c + c1 region (550 nm) is depressed relative to that in the cytochrome b region (562 nm). It appears that formation of all cytochromes, particularly cytochrome c, is depressed sufficiently to limit aerobic growth of small-type Q2 cells (Table 5). However, when growth medium is supplemented with Alv, synthesis of all cytochromes is markedly enhrtnced (trace B, Fig. 4) and growth limitation disappears. Examination of spectra, of large-type Q2 cells gave the surprising result illustrated by traces C and D. Cyto. chrome synthesis in this substrain is also enhanced by Alv supplementation. Apparently, then, the two substrains both contain the mutant cyd gene, but only in the small type is the defect expressed sufficiently to cause appreciable limitation of growth. The genetic basis for this variability between substrains is currently undel investigation. Cytochrome spectra of the normal yeast strain, D311-3A, were not, detectably changed when growing cells were supplemented with Alv. One of the small-type clones, designated &2(Ml) was chosen for subsequent work, as described below. (j) Absence of cyd in strain &I p+ It may be noted from Figure 2 that the spectra of strain Ql (p + ) give no indication of a cytochrome deficiency. Moreover, when an attempt was made to reproduce the results given in Table 1, no cytochrome c deficient petites were produced from strain Ql at either concentration of ethidium bromide, although they were again formed from strain Q2. Furthermore, no evidence could be found that the original strain Ql contained a mixed population of cyd p+ and CYD p+ cells. Cells from an old Ql culture, which had been made before the isolation of the cytochrome-deficient mutants (Table I), were plated to form single colonies. Small-type clones were invariably p- and contained normal amounts of cytochrome c, while large-type clones were always p+. None of the subclones was affected significantly by Alv. Although a cyd gene pre-existed in strain QS p +, the cyd gene in strain QlCYD-, must have arisen by a chance mutation which occurred during the original ethidium bromide treatment (Table 1). (k) l+.mtionul

allelim

of independently derived cyd genes

Although some GT5 segregants derived from the cross QlCYD-, x D311-3A produced CYD- petites upon treatment with ethidium bromide (Table 6), the possibility remained that the p + forms of these segregants, like strain Ql, did not actually contain a cyd gene, but were instead specifically susceptible to ethidium bromideinduced mutation to CYD -. However, when cells of the p+ segregants GT5-1C and GTB-1D were grown with and without added Alv and then examined in the spectroscope, an enhancement of all cytochromes by Alv was observed. These strains are therefore phenotypically similar to strain Q2 (p+ ). The Alv-induced changes in the

30

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ET

AL.

TABLE 7 EJects of &aminolevulinic acid addition on mmzal and cytochrorne c-dej%ent mutants stmin

Genotype

no.

D311-3A GT6-1A D624-2D D283-SD D286-1C D304-SD D332-4A B271 (L) B271 (S) D293-1A D312-1D

Ql GTS-1C GT6-1D GT6-SD

Q2 (Ml) GT6-13D GT6-6D-CYD

-1

Normal (CYD) Normal cycl-1 yc2-1 yc3-3 cyc3-3 yc3-6 cycl-1 cyc4- 1 cyc6-1 cyc6-1 Normal cydl-1 ydl-1 cydl-1 ydl-2 cydl-2 Ydl-1 b-1

Cytochrome No Alv 6 7 2 2 2 2 4 6 2 6 6 7 6 3 4 3 2 1

c units + Alv I 7 2 2 2 2 4 7 6 6 6 7 7 7 7 7 7 7

Cytochrome c concentration of cells frozen in liquid nitrogen was estimated visually using a spectrosoope. Key to units: 1, barely detectable; 2, very low; 3 and 4, low; 6, slightly deficient; 6 and 7, normal renge. Cells were grown at 30°C for 48 h on Petri dishes of dextrose agrtr medium. Straina B271 (L) ad B271 (S) represent two separrtte clones isolated from a large colony (L) and a small colony (S), respectively, after plating the original strain on dextrose agar medium. Alv ooncentration was 0.6 rnw.

cytochrome c concentrations of these p + segregants are shown in Table 7. The smalltype Q2 substrain, Q2(Ml), and a p+ segregant, descended from Q2, GT6-13D, show comparable changes. On the other hand, the p+ segregant GTB-lA, which produced only typical p- cells when treated with ethidium bromide, had a normal cytochrome c content, which was unaffected by Alv supplementation. Similarly, Alv had no significant effect on cytochrome c production in strain Ql or in the normal control strain D311-3A. The enhancement of the cytochrome c deficiency by mutation from p+ to p- is also found among the GT5 segregants, as illustrated with strain GT6-5D. When the p+ form was tested, four units of cytochrome c were observed, but after ethidium bromide treatment and isolation of the petite (p-) form, GT5-5D-CYD-,, cytochrome c absorption was barely detectable in the spectroscope (1 unit). Therefore, these results indicate that the genotype of such GT5 segregants may, like strain Q2, be designated by cyd p+ . The marked similarity in phenotype of the independently derived cyd mutants naturally raises the question of their possible functional allelism. To test this possibility, complementation tests were carried out, as shown in Table 8. Strain QICYD-, was crossed with the segregant GT6-lC, which was descended from strain Q2CYD-,. The resulting diploid, GTlO, was then grown in the presence and absence of 0.5 mmAlv, and as shown in Table 8, cytochrome synthesis was greatly increased by Alv.

6-AMINOLEVULINATE-DEFICIENT

31

YEAST

TABLE 8

Functional allelism of cyd genes Diploid no.

Cytochromo No Alv

Strains

Ql QlCYD-,

QWfl)

GT21 GTlO GT22 GT23 GT9 GT24 GTll Cytochrome

Q2CYD-, CT&SD GTB-IC QlCYD-, x GTB-SD QICYD-8 x GTG-1C CT&SD x QZCYD- 1 Ql x GTS-SD Ql x GTG-1C GT6-6D x &2(Ml) GTG-1C x Q2(Ml) c was estimated

visually

c units +Alv

7

7

1 3 2 4 3 2 2 2 6 7 3 3

G 7 6 7 6 6 6 6 G 7 6 7

with a spectroscope,

Genotype

cydl-1 cydl-1 cydl-1 CYD CYD cydl-1 cydl-2

as indicated

CYD cydl-1 cydl-2 cydl-2 cydl-1 cydl-2 p- x p- x p+ x p+ x p+ x

p+ p+

P+

pp+ p-

p+

p+ cydl-1 p+ cydl-2 p+ cydl-2 pcydl-1 p+ cydl-2 p+ x cydl-2 p+ x cydl-2 p+

in the legend to Table 7.

Therefore, we may conclude that GTlO is homozygous for a cyd gene, that is, the cyd genes in QlCYD-, and GTG-IC are functional alleles, and may be designated cyd 1- 1 and cyd l-2, respectively. This conclusion is confirmed by the behavior of diploid strains GT22, GT24 and GTll which show that the cyd determinants in strain &2(Ml) and selected segregants of different descent are allelic. Diploid strains GT9 and GT23, in contrast, contain normal concentrations of cytochromes which are unaffected by Alv supplementation. This result is consistent with the previous conclusion that strain Ql (p+ ) contains a normal CYD gene. (1) Comparison of cyd-1 strains with known cytochrome c-defiient mutants It was pointed out that cytochrome c in small-type Q2 (p’) is particularly deficient, as indicated by the spectrum in Figure 4, trace A, and by the data in Table 8, line 3. In this respect this strain resembles several of the cytochrome c-deficient cyc mutants (formerly designated cy) described by Sherman (1964). Moreover, it has also been observed that destruction of p factor strongly enhances the expression of cytochrome c deficiency in cetiain strains containing either a cyc-2 or a cyc-3 gene (Sherman et a.!., 1965). It was of special interest, therefore, to compare various cyc mutants with the cyd-1 strains. Two types of experiments were done. In the tirst, diploids were constructed, making all possible pairwise crosses between haploid strains containing cydl-1, and tester strains each containing a different cyc mutant gene (cyc-1 through cyc-6). A similar set of crosses was made using cydl-2 strains and all the different cyc testers. Appropriate control diploids were also prepared by crossing each type of eyd or cyc strain with an appropriate wiId-type tester. All diploids were then grown on unsupplemented dextrose medium, and the cytochrome content of each cell type was estimated with the spectroscope. In the second type of experiment, all the haploid strains,

H.

32

K.

SANDERS TABLE

ET

AL.

9

Complemenkztion tests with cytochrome-dejicieti and cytochrome c mutanls Diploid no. GT26 CT12 CT14 GT16 GT27 CT28 CT20 CT29 GT30 GT31 CT32 GT33

CYC gene l-l 2-1 3-3 3-3 3-5 3-5 4-1 4-l 4-l 5-1 6-l 6-1

Tester no. QlCYD-s QlCYD-s QlCYD-, QlCYD-s GTB-13D GTS-1C GTG-1C GT6-1C-CYD-1 GTS-1C GTS-SD-CYD - 1 QlCYD-s Q2CYD-1

Tester genotype cydl-1 pcydl-1 pcydl-1 pcudl-1 pcydl-2 p+ cydl-1 p+ cydl-2 p+ cydl-2 pcydl-1 p+ cyczl-1 pcydl-1 pcydl-2 p-

Cytochrome No Alv 6 6 6 6 6 4

c units +Alv 6 7 6 6 6 6

7

7

6 4 6 6 6

7 6 6 6 6

Cytochrome c was estimated visually with a spectroscope &s indicated in the legend to Table 7. The cyc3-3 strains used in preparing GT14 8nd GT16 were D286-1C and D304-6D, respectively. Cytochrome c units for haploid strains are shown in Tables 7 and 8.

each containing one of the cyc genes, and the various (cyc x cyd) diploids were tested for their response to Alv supplementation. Representative results are presented in Tables 7 and 9. As indicated in Table 7 each of the cyc p+ strains except the cyc5-1 strain exhibited some degree of cytochrome c deficiency. However, only strains containing the cyc4-1 gene show a detectable response to Alv. The (L) and (S) forms of strain B271 represent substrains isolated respectively from large and small colonies formed by spreading a dilute suspension of B271 cells on dextrose medium. Both are p + . These results indicate that neither cyc-2 nor cyc-3 is allelic to cyd-1, notwithstanding similarities in response to p factor destruction (Sherman et al., 1965). Likewise, the cyc-6 gene and the cycl-1 gene appear to be functionally different from cyd-1. Since cycl-1 is known to be the structural gene for yeast iso-1-cfiochrome c (Sherman et al., 1966) the result with this strain was not unexpected. Confirmation that the various cyc genes differ from cyd-1 was obtained from the complementation study illustrated in Table 9. Diploids heterozygous for each of the six cyc genes and for cyd-1 were found to produce normal amounts of cytochrome c even in the absence of Alv. Although three diploids, GT28, CT30 and GT33, showed a small degree of cytochrome c deficiency, these apparently do not indicate functional allelism, since similar diploids constructed with the same cyc genes but different cyd tester strains contained normal concentrations of cytochrome c. This suggests that the background genomes of some strains heterozygous for cyd-1 permit a partial expression of the cyd mutant phenotype, since in these particular diploids, Alv restores the cytochrome c concentration to normal. Of particular interest is the observation that complementation takes place between cyc4-1 and cyd-1 strains. This indicates that cyc-4 and cyd-1 represent different genes. In order to exclude the possibility of intragenic oomplementation, the linkage test

S-AMINOLEVULINATE-DEFICIENT

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33

TABLE 10 Test for linkage between cydl-2 and cyc4-1 genes Type of tetrad

Response to Alv (+):(-)

Parental ditype Non-parental ditype Tetratype

4:o 2:2 3:l

No. of tetrads of type 3 3 9

Segregants derived from diploid strain GT20 (Table 9) were grown at 30°C for 48 h on dextrose agar medium with and without 0.6 mma-Alv supplement. Presence (+) of a cyd-1 of cyc-4 gene (or both) was indicated by an Alv-dependent increase in cytochrome c similar to those given in Table 7. Absence (- ) of these genes was indicated when cytochrome concentrations were in the normal range and were unaffected by Alv.

summarized in Table 10 was done. The results show clearly that cydl-2 and cyc4-1 are distinct unlinked genes which produce a defect in an early step in porphyrin biosynthesis. (m) 6-Aminolevulinic

acid-induced changes in mitochondria isolated from cyd p+ cells

The presence of the cyd-1 mutation in p + strains causes a partial deficiency in the cellular concentration of all cytochromes (Fig. 4). At the level of mitochondria there are certain possible situations which might be responsible for this : (1) the number of mitochondria per cell may be less than normal. (2) Mitochondrial composition may have changed such that the inner membrane is relatively deficient in all cytochromes. (3) Both number and composition may be different from normal. In an effort to obtain information concerning these possibilities, strain Q2 (cyd-1 p+) was grown on a large scale with and without added Alv, and intact mitochondria were isolated from each cell type. Both kinds of mitochondria oxidized succinate or ethanol and exhibited

Band I

Bond 2 -----_

- Alv

i Air

Pm. 5. Changes in sedimentation behavior of cyd mitochondria induced by growing strain Q2 p+ in medium containing Alv. Cells were grown at 30°C in dextrose medium in a fermentor. The Alv concentration used was 0.6 mM. Mitoohondria were isolated by differential sedimentation before placing them on gradients. Linear Renografln (20% to 40%) gradients were used. 3

34

H. K. SANDERS

ET AL.

reversible respiratory control in response to small pulses of ADP (Balcavage 8s Mattoon, 1968). Specific respiratory rates and respiratory control ratios of mitochondria from unsupplemented cells were only slightly lower than those of organelles isolated from supplemented cells. However, as shown in Figure 5, the two types of mitochondria behaved quite differently on Renografin gradients. Mitochondria from unsupplemented cells banded higher in the gradient, and small, variable amounts of material banded at the top of the gradient. These distinct differences in sedimentation behavior suggest that the two types of mitochondria differ significantly in composition. Possible differences in lipid content are suggested, and are currently under study. Size changes may also contribute to the differences. (n) Effect of S-aminolevulinic acid precursors on growth of cyd-1 and cyc4-1 strains

A cytochrome-deficient yeast mutant studied by Raut (1953) and by YEas & Starr (1953) was shown to be deficient in porphyrin synthesis as a result of partially defective glycine biosynthesis. Neither cyd-I nor cyd-1 mutants exhibited more than a small enhancement in cy-tochrome synthesis in minimal medium supplemented with 5 rnM-glycine, 5 mM-glutamate and 4-9 PM-pyridoxine hydrochloride. Presumably added glutamate could be converted to succinyl CoA, an Alv precursor, by transamination and oxidative decarboxylation. This result indicates that cytochrome synthesis in these mutants is limited by the supply of Alv rather than by the supply of one of its precursors or of the Alv synthetase cofactor, pyridoxal phosphate.

4. Discussion The isolation of yeast mutants dependent upon added Alv for normal cytochrome biosynthesis should be of particular interest to investigators studying the regulation of mitochondrial biogenesis. Because the effective intracellular concentration of Alv represents a primary controlling factor in synthesis of all respiratory cytochromes, the cyd mutants may serve as an experimental system in which mitochondrial development may be manipulated directly, simply by altering the concentration of added Alv. This new experimental system is particularly well-suited for gaining information GompIementary to that obtained in the wideIy studied experimental porphyrias (Tschudy & Bonkowsky, 1972). The yeast system, moreover, has the special advantage that it may be readily subjected to additional, controlled genetic manipulation. This may be accomplished by breeding or mutation to introduce modified nuclear genes or genes residing in mitochondrial DNA. The unmasking of the cyd gene caused by mutation from p + to p- provides a dramatic example of this type of secondary genetic modification. It is not possible, from the results presented here, to determine the exact site of the mutant lesion. Although it is tempting to conclude that cyd-1 represents an altered structural gene for Alv synthetase, other possibilities may be considered. Current evidence obtained with porphyria animals indicates that Alv synthetase is first produced by cycloheximide-sensitive translation in the cy-tosol, then incorporated into the matrix of mitochondria (Beattie & Stuchell, 1970), as indicated in the scheme presented in Figure 6. Moreover, in animal tissues Alv synthetase has a very much shorter half-life (Tschudy & Bonkowsky, 1972) than most mitochondrial

8..AMINOLEVULINATE-DEFICIENT NUCLEUS

YEAST

35

1

Mitochondrial gene product

AIV synthetose

Fro. 0. Hypothetical regulatory network showing participation of both nuclear and mitochondrial genetio information in control of Alv synthetaee. Various alternatives are shown involving either direct interaction of gene products or indirect regulation by low molecular weight metabolites. Various possibilities are discussed in the text.

proteins (Ashwell & Work, 1970), indicating that it undergoes rapid degradation. It is likely that each of these three processes, enzyme synthesis, incorporation into mitochondria and enzyme degradation, is subject to genetic regulation. While some aspects of regulation may be predetermined in the primary stru&i.re of the Alv synthetase itself, other regulatory genes may also participate. The present demonstration that either of the two complementary genes, cyd-1 and cyc-4, produce an Alv deficiency constitutes evidence that at least two different genes are involved in maintaining normal Alv production. Conceivably one of these mutations affects the primary structure of Alv synthetase while the other interferes with some aspect of regulation. In this connection it may be noted that Whiting & Elliott (1972) have recently shown that purified Alv synthetase isolated from liver mitochondria differs in molecular weight from an enzyme isolated from liver cytosol. However, since the two enzymes have similar immunological specificity, these authors seem to favor the view that the cytosol enzyme is a precursor of the mitochondrial enzyme. The kinetic experiments of Beattie & StucheU (1970) support this view. Since the molecular weight of the cytosol enzyme (178,000) is much larger than that of the mitochondrial enzyme (77,000), it is likely that incorporation of Alv synthetase into mitochondria requires some type of processing, possibly involving enzymatic cleavage of a peptide bond in a larger cytoplasmic “pro-enzyme”. It has also been suggested that the mitochondrial enzyme is attached to the inner surface of the inner mitochondrial membrane by disulfide bonds, since its solubilization required both high salt and a reducing agent, dithioerythritol (Whiting & Elliott, 1972). Such an attachment might also be enzyme catalyzed. A further possibility for enzymatic processing of Alv synthetase might involve detachment of the synthetase from the mitochondrial membrane and its subsequent degradation. Any one of these proposed enzymatic steps may be invoked to explain the existence of two nuclear genes controlling Alv synthesis. The unmasking of the latent cyd-1 defect by mutation from p+ to p- reveals an underlying relation between information coded in mitochondrial DNA and some

36

H. K. SANDERS

ET AL.

aspect of porphyrin synthesis. The exact nature of this relation is not evident from the results obtained so far, but certain possibilities may be considered. Since there is general agreement that the products of intramitochondrial protein synthesis are components of the inner mitochondrial membrane (Borst, 1972), it may be suggested that one or more of these components participates in the processing of Alv synthetase as it is introduced into the mitochondrion. However, it must be emphasized that destruction of mitochondrial DNA is not, by itself, sufficient to inhibit porphyrin synthesis, since most p- strains contain high concentrations of cytochrome C, and frequently accumulate porphyrin (Pretlow & Sherman, 1967). Although Alv synthetase appears to function normally, either within the incomplete organelles of p- strains (Labbe, 1971; Petzuch, 1971), or possibly within the cytosol, it seems reasonable to expect that the environment of the enzyme in p- cells differs significantly from that in p+ cells. Moreover, it is likely that there are important differences in the processing of newly-formed enzyme (or pro-enzyme) in a p- strain. While normal Alv synthetase retains activity in spite of such postulated differences in p- cells, an abnormal (cyd-1) enzyme might well be inactivated when exposed to a similar stress. If in fact the gene product of cyd-1 (or cyc-4) is Alv synthetase, it should be possible to produce mutations at the same locus which result in complete loss of Alv formation, even in P+ cells. However, it might be necessary to add Alv to the screening medium in order to detect such “non-leaky” mutants. As an alternative to this hypothesis, one could propose that some unspecified aspect of the regulation of Alv synthesis is controlled by the joint action of a mitochondrial (p+) gene and a nuclear gene (CYD1). The normal allele of either gene is sufficient for continued Alv production, but mutation in both genes results in almost complete loss of Alv synthesis. Regardless of the mechanism responsible for the unmasking of cyd by the pmutation, the significance of the observation is clear : the expression of a nuclear gene controlling a key reaction in mitochondrial biogenesis is highly dependent upon the presence of a cytoplasmic (mitochondrial) gene, For such control to function, some intracellular exchange of information between the two genetic compartments must occur. Whether this communication occurs at the macromolecular level or involves a movement of low molecular weight metabolites or regulatory molecules remains an open question. Similar nuclear-cytoplasmic interactions have previously been observed. In a previous report from this laboratory (Beck et al., 1968) mutation to p- was found to be lethal to yeast containing the nuclear gene, pet-9 (ps), which, in p+ strains, reduces the affinity of mitochondria for ADP. As in strains containing cyd-1, mutation in mitochondrial DNA (to p-) appears to unmask an underlying defect which, in the case of pet-g, is potential lethality. Similar indications of nuclear-mitochondrial communication were found by Sherman et al. (1965) in cytochrome c-deficient mutants containing either cyc-2 or cyc-3 genes. When the relative concentrations of cytochrome c in p+ and p- forms of these mutants were measured, p- strains were found to have much lower concentrations. Most extreme in this respect were cyc3-3 p- strains which contained less than 20% as much cytochrome c as the corresponding cyc3-3 p+ strain. The striking similarity between the cyd mutants ad the cyc-2 and cyc-3 strains, pointed out to us by Dr Sherman, led us to make a careful comparison between eyd mutants and the various cyc strains. Although the complementation results indicate that none of the six cyc strains is allelic to cyd-1, some marked resemblances were found. In the fist place, it is shown that while all cytochromes are partially deficient

S-AMINOLEVULINATE-DEFICIENT

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37

in cyd-I pi strains, the deficiency in cytochrome c is most conspicuous. This phenotypic trait, by itself, observed under different circumstances probably would have led us to classify the strains as cytochrome c-deficient (cyc). Another notable similarity is the response of cyc4-1 to added Alv. Since the deficiency in cytochrome c production in this strain may be restored to normal by added Alv, this mutant could be more accurately classified as porphyrin-deficient, rather than cytochrome c deficient. The key feature of these similarities is that partially deficient porphyrin biosynthesis in p+ strains results in greater relative deficiencies in cytochrome c than in other cytochromes. This observation has two important implications. First, it suggests the possibility that some of the cytochrome c-deficient strains with mutations at various loci may represent mutants partially deficient in different enzymes of porphyrin biosynthesis, Second, it indicates that when porphyrin biosynthesis becomes limiting, a differential in the apparent rates of synthesis of the individual cytochromes is imposed. One possible interpretation of this differential is that porphyrin (or heme) destined for incorporation into cytochrome c must be exported from mitochondria, while that destined for producing cytochromes a and b is retained in the organelle. According to this idea, conversion of apocytochrome c to holoenzyme would occur in the cytosol, while the heme incorporation into cytochromes a and b would be intramitochondrial. Presumably, then, the greater relative restriction in formation of cytochrome c would indicate that the exportation process was less efficient than the intramitochondrial process (see Fig. 1). Previous reports of S. cerevisiae mutants deficient in all cytochromes have appeared. A Mendelian mutant exhibiting no cytochrome absorption bands except for a very faint cytochrome c band was isolated by Raut (1953). A strain containing this mutation was shown by YEas & Starr (1953) to be a glycine auxotroph which produced only enough glycine for limited growth. When this strain, which is also p - , was supplemented with either glycine or protoporphyrin IX, synthesis of cytochrome c and catalase was restored. This mutant therefore differs from the cyd-1 p- strains, which exhibit little or no increase in cytochromes in response to glycine supplementation. Sugimura et al. (1966) have also described a cytochrome-deficient mutant. Like the glycine auxotroph just discussed, this mutant lacked catalase and the defect was determined by a single nuclear gene (Gunge et al., 1967). Moreover, hemoprotein synthesis could be restored by addition of protoporphyrin IX (Miyake & Sugimura, 1970). This mutant appears to have a defect in one of the later stages of porphyrin biosynthesis, since it accumulates large amounts of coproporphyriu and some u.roporphyrin, and whole cell spectra are characterized by strong absorption bands at 538 and 575 nm. In this respect, then, it differs from cyd-I, which does not exhibit any significant absorption bands. Since cyd-1 strains do not respond appreciably to glycine, pyridoxine or glutamate, display no porphyrin accumulation, but do respond to Alv, it is quite likely that the defect in these mutants involves the activity of Alp synthetase, its biosynthesis, or its incorporation into mitochondria. Direct confirmation of this hypothesis awaits studies at the enzymatic level. Since one can significantly alter the cytochrome concentration of intact cyd p+ cells by changing the Alv concentration of the growth medium, it was of considerable interest to determine the basis of this change at the level of the mitochondria themselves. If the primary effect of Alv supplementation is to increase only the number of mitochondria per cell, one might suggest that information indicating activity of the

H.

38

K.

ET AL.

SANDERS

normal C YD-1 gene is communicated directly to mitochondrial DNA. This information, possibly transmitted in the form of a porphyrin metabolite, would then act as a signal to initiate replication and transcription of mitochondrial DNA. However, the significant difference

in behavior

on Renografin

gradients

of mitochondria

isolated

from

supplemented and unsupplemented cyd-1 yeast suggests that the composition of the mitochondria is subject to regulation by the Alv supply. This information indicates that change in numbers of mitochondria may not, by itself, account for the change in cellular cytochrome content. It follows then, that the diverse biosynthetic processes required for mitochondrial biogenesis (synthesis of lipids, proteins, nucIeic acids, and cofactors) are not all stringently controlled by the first committing step in porphyrin synthesis, in spite of the fact that this reaction, by its very nature, can exert simultaneous

control

over synthesis

of all of the mitochondrial

cytochromes.

The type of

changes in composition responsible for the observed behavior of the mitochondria Renografin gradients is currently under study.

in

This investigation was supported by grants from the National Institutes of Health (GM15884) and the North Atlantic Treaty Organization. The authors are indebted to Karan Sapp for excellent technical assistance and to Linda Fraula for her help in preparing the manuscript. REFERENCES Ashwell, M. & Work, T. S. (1970). Annu. Rev. Biochem. 39, 251. Balcavage, W. X. & Mattoon, J. R. (1968). Biochim. B~@hys. Acta, 153, 621. Balcavage, W. X., Beck, J. C., Beck, D. P., Greenawalt, J. W., Parker, J. H. & Mattoon, J. R. (1970). Cryobiol. 6, 385. Beattie, D. S. & Stuchell, R. N. (1970). Arch. Biochem. Btiphys. 139, 291. Beck, J. C. & Mattoon, J. R. (1971). Genetics, 68, S4. Beck, J. C., Mattoon, J. R., Hawthorne, D. C. & Sherman, F. (1968). Proc. Nat. Acud. Sci., U.S.A. 60, 186. Beck, J. C., Parker, J. H., Balcavage, W. X. & Mattoon, J. R. (1971). In Autonomy and Biogenesis of Mitochondria and Chloroplasts (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds), p. 194, North-Holland, Amsterdam. Borst, P. (1972). Annu. Rev. B&hem. 41, 333. Dawid, I. B. (1972). J. MOE. Biol. 63, 201. Ephrussi, B. (1953). In Nucleo-cytoplaamic Relations in Microorganiems, p. 13, Clarendon Press, Oxford. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D. R. & Marmur, J. (1970). J. Mol. Biol. 52, 323. Gunge, N., Sugimura, T. t Iwasaki, M. (1967). Genetica, 57, 213. Hawthorne, D. C. BEMortimer, R. K. (1960). Genetk, 45, 1085. Hayashi, N., Yoda, B. & Kikuchi, G. (1969). Arch. Biochem. Biophys. 131, 83. Labbe, P. (1971). Biochimie, 53, 1001. Mattoon, J. R. t Sherman, F. (1966). J. Bd. Chem. 241, 4330. McKay, R., Druyan, R., Getz, G. S. & Rabinowitz, M. (1969). B&hem. J. 114, 456. Miyake, S. & Sugimura, T. (1970). Biochem. Biophys. Res. Commun. 40, 85. Mortimer, R. K. & Hawthorne, D. C. (1969). In The Yeasts (Rose, A. H. & Harrison, J. S., eds), vol. 1, p. 386, Academic Press, New York. Nagley, P. & Linnane, A. W. (1970). B&&em. Biophys. Re8. Comnau% 39, 989. Parker, J. H. & Mattoon, J. R. (1969). J. Bacterial. 100, 647. Petzuch, M. (1971). C.R.H. Acad. Sci. 273, 105. Pretlow, T. P. & Sherman, F. (1967). Biochim. Biophys. Acta, 148, 629. Raut, C. (1953). Exp. Cell Re-s. 4, 295. Sano, S. & Granick, S. (1961). J. Bid. Chem. 236, 1173. Scholnick, P. L., Hammaker, L. E. & Marver, H. S. (1969). Proc. Nat. Acad. SC~., U.S.A. 63, 65.

6-AMINOLEVULINATE-DEFICIENT Sherman, Sherman, Sherman, Sherman, Sherman,

F. F. F. F., F.,

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(1964). Genetics, 49, 39. & Ephrussi, B. (1962). Genetics, 47, 695. & Slonimski, P. P. (1964). Btichim. Biophys. Acta, 90, 1. Taber, H. & Campbell, W. (1965). J. Mol. Biol. 13, 21. Stewart, J. W., Margoliash, E., Parker, J. & Campbell, W. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 1498. Sugimura, T., Okabe, K., Nagao, M. & Gunge, N. (1966). Biochim. Biophys. Actu, 115,267. Tschudy, D. P. & Bonkowsky, H. L. (1972). Fed. Proc. Fed. Amer. Sot. Ezp. Biol. 31, 147. Whiting, M. J. & Elliott, W. H. (1972). J. Biol. Chem. 247, 6818. Wintersberger, E. (1967). 2. Phytiol. Chem. 348, 1701. YEas, M. & Starr, T. J. (1953). J. Bacterial. 65, 83.