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Glucose repression of cytochrome a synthesis in cytochrome-deficient mutants of yeast
54o
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95165
GLUCOSE R E P R E S S I O N OF CYTOCHROME a S Y N T H E S I S IN C Y T O C H R O M E - D E F I C I E N T MUTANTS OF YEAST
CONOR REILLY ANDFRED SHERMAN Department o[ Radiation Biology, University o/Rochester School o[ Medicine and Dentistry, Rochester, N.Y. (U.S.A.) (Received September 2rid, 1964)
SUMMARY I. Cytochrome content and respiratory activities were investigated in normal and cytochrome-deficient mutants of yeast which were under various degrees of catabolite (glucose) repression. 2. Derepression could be obtained b y growing yeast in melibiose, probably due to the low rate of utilization of this felmentable sugar. 3. The synthesis of cytochrome a (i.e., cytochromes a and aa) was very sensitive to catabolite repression in single-gene mutants (cya_ 5 and cy3_~) and a doublegene m u t a n t (cyl_lcys_3), which were partially deficient in cytochrome c, and in a single-gene m u t a n t (Pl0), which was deficient in cytochromes b and c1. Cytochrome a synthesis behaved normally in other cytochrome c deficient mutants (cYl-1, cy~-l, and cy34 ). In some strains, cytochrome a synthesis was completely blocked. 4- I t is suggested that the synthesis of cytochrome a is normally regulated by other cytochromes.
INTRODUCTION Normal, aerobically-grown Saccharomyces cerevisiae has a classical cytochrome system, containing cytochromes a, b, cl, and c. In spite of the fact that these cytochromes are distinct and separable proteins 1, certain single-gene mutants ~ and cytoplasmic mutants 3 have been found to have multiple deficiencies 4. Although a number of tentative theories have been presented 4, no clear explanation has been given to account for a single gene controlling more than one cytochrome. A large number of cytochrome-deficient mutants have been iecently isolated b y a new procedure 5. The present investigation of the cytochrome content of some of these mutants has revealed the dependence of the synthesis of cytochrome a (i.e., cytochromes a and c~) on the presence of other cytochromes. The synthesis of Abbreviations used for culture media: Y, I ~O Bacto-yeast extract; P, 2 °/o Bactopeptone; D, dextrose (with percentage indicated); M, melibiose (with percentage indicated); G, glycerol (with percentage indicated). Biochim. Biophys. Acta, 95 (1965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
641
cytochrome a, in certain cytochrome c-deficient mutants and cytochrome bdeficient mutants, has been found to be extremely sensitive to glucose repression.
MATERIALS AND
METHODS
Nomenclature
The original symbols~, 6 used to designate m u t a n t characters controlling the cytochrome and related systems are: p, for single-gene mutants which are unable to grow on nonfermentable substrates (e.g. glycerol, lactate, ethanol and acetate) as the sole carbon source, and which m a y or m a y not have an altered absorption spectrum; cy, for single-gene mutants having an altered absolption spectrum (e.g., the loss of a cytochrome) but still retaining the ability to utilize nonfermentable carbon sources; and p - f)r cytJplasmic mutants a which are unable to utilize nonfermentable carbon sources. The symbols employed to desiglaate these wild type characters are: P C Y p+. However, the terminology is somewhat misleading since, not only is an artificial distinction made between cy and p mutants, but independent mutants at the same locus can have either a p or cy phenotype. For example, cy3_1, cya-3, cY~-5, and cyg_6 are all mutants at the same locus, but cy3_ ~ and cyg-n can utilize nonfermentable carbon somces, while cy3-5 and cy3-8 cannot. Also m a n y cy mutants have a pronounced decrease of growth rates in synthetic media containing certain nonfermentable substrates as the sole carbon source We will, however, retain the original symbols as previously published.
Strains and genetics
The origin and genetics of the m u t a n t genes cyl_ 1 (ref. 7), cy3-1, cy~-3, and md-cyg_ ~ (ref. 5) have been described. B-3o6 (Pa0) was induced b y nitrous acid and detected b y the spectroscopic method s. In a cross to a normal strain, Pl0 segregated as a single-gene in five asci. The method of detecting cy3_5 and cy3_~, as well as other cytochrome-deficient mutants, will be described elsewhere, gltl_ 1 (strain number 37892) was kindly given to us by Dr. M. OGURs.
Culture methods
Liquid growth media contained 1 % Bacto-yeast extract, 2 % Bacto-peptone, and various concentrations of different carbon sources which are indicated as follows: Y P 2 % D , 2 % dextrose; Y P I % D , 1 % dextrose; Y P I % M , 1 % melibiose, etc. Two percent agar was added in preparing solid media. Freshly grown cells were inoculated to lO 6 cells/ml and incubated at 3 °0 with vigorous shaking. In order to assure good aeration, the erlenmeyer growth flasks were never filled to more than IO % of their total volume. Unless stated otherwise, cells were harvested at early stationary phase of growth. 13iochim. Biophys. Acta, 95 (1965) 640-651
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C. REILLY, F. SHERMAN
Growth rates were determined with 125 ml Nephelo-culture flasks containing io ml of media. The absorbancy of cultures was measured at 45 ° m/z with a Spectronic 20 spectrophotometer (Bausch and Lomb). Special reference standards were employed in order to measure the entire growth curve without diluting the culture at high densities.
Respiration Respiration rates of intact cells were determined by the direct Warburg method at 3 °° in the presence of air. 2,0 ml of reactive material contained: o.i M glucose; 67 mM KH2PO 4 at p H 4.6; and 2.5 mg (dry wt) of yeast. 0.2 ml of 20 % KOH was added to the center well and sometimes to the side arm when utilizing strains having very low rates of oxygen consumFtion. Qo2 was expressed as #1 of oxygen consumed per mg dry wt. of yeast per hour.
Cytochrome spectra The cytochrome content of intact cells was estimated by low-temperature (--19 o°) spectroscopy by the method previously described by SHERMAN AND SLONIMSKI4. In some instances, low-temperature recordings were also made 6.
Homogeneity of cell populations Obviously, relatively homogeneous cell suspensions should be used for measurements of respiration and cytochrome content. The frequencies of backmutants in mutant strains, which are unable to grow on nonfermentable carbon sources, can be assayed by simply plating the cell suspension and determining the proportion of cells giving rise to colonies on YP3 %G (I % yeast extract, 2 % peptone, and 3 % glycerol). In all strains employed in the present study, the proportion of backmutants was negligible except for strain B-3o6 (Pl0), which had from o to 25 % in various experiments. In these cases corrections have been made as indicated below. In the study of cytochrome mutants, one possible source of error is the relatively high frequencies of p p- cells found in some p p+ cultures ~. Although the frequency of p- cells can be easily determined in normal strains by the triphenyltetrazolium chloride-overlay method 9, or by plating on YP o. I°/oD3°/o G (I % yeast extract, 2 % peptone, o.I °/o dextrose, and 3 % glycerol), it is more difficult to' make such determinations with certain mutant strains. Two methods have been employed for determining the exact frequencies of p- cells in mutant cultures: (I) When zygotes from a cross of the mutant with a neutral vegetative mutant are plated, only the p p- cells form p- hybrids 1°. (2) Sometimes the pigment produced by adI (adenine dependence) mutants is affected by the cytochrome phenotype so that p p+ and p pcolonies exhibit distinct colors1°, 11. In the investigation presented herein, we have developed still another method which has certain obvious advantages. Approx. 5O-lOO cells, from the haploid population to be tested, were plated Biochim. Biophys. Acta, 95 (I965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
643
per YP2~oD plate and the plates incubated for about three days until the colonies were formed. A second plate was then prepared by streaking approx, lO8 cells per plate of a P p- strain of the opposite mating type. The first plate, which contained the colonies to be tested, was replicaplated on the second plate, and the mating mixture incubated for one day. This plate was either stained directly with triphenyltetrazolium chloride, or replicaplated on Y P 3 % G . In both cases, the frequency of p- ceils in the original population can be inferred from the frequency of p- hybrid colonies. The frequencies of p- cells in all normal and mutant strains were determined either directly by plating on YP o. I ~o D3 ~o G (in cases when the strain in question could utilize nonfermentable carbon sources) or indirectly by the mating method described above. In all cases, the frequencies of p- cells were negligible, constituting less than IO % of the cell populations.
RESULTS
Characteristics o[ normal yeast grown with various carbon sources
Aerobic growth of normal strains of yeast in high concentrations of glucose results in lowering of the Qov cytochromes and related enzymes, and mitochondrial formation 12-17. It is well known that glucose repression of the cytochrome system can be alleviated by growth in nonfermentable carbon sources. However, as previously pointed out ~, this method cannot be employed to derepress certain mutants which are unable to utilize nonfermentable substrates for growth. It is possible to circumvent this condition by growing the mutants in limiting glucose, as in a chemostat, or by feeding a limiting amount of glucose to the growing culture. Since these procedures require special equipment and are cumbersome for the study of large numbers of strains, we have investigated conditions which simulate low concentrations of glucose, and still produce a high yield of cells. It was reasoned that the catabolism of certain fermentable substrates could be limiting, due to low concentrations of hydrolyzing or other enzymes. With this goal in mind, a normal yeast strain (D273loB) was grown in a number of media containing various fermentable sugars, and a nonfermentable carbon source (ethanol). As shown in Table I, the completely derepressed strain, which was grown in ethanol, had a Qo2 of approx. 15o. The same rate of respiration could be obtained after growth in melibiose. No decrease in catabolite (glucose) repression was observed after growth in sucrose or galactose (Table I). Other carbohydrates (mannose, trehalose, melezitose, and starch) either could not be utilized by the yeast, or also failed to diminish catabolite repression. As will be discussed below, an increase of oxidative activity was also observed after growth in raffinose (Table I). It was therefore concluded that melibiose is the best fermentable sugar to use for obtaining derepressed yeast. In retrospect, the same reasoning can explain the early observation of STRITTMATTER13 that a specific galactose-grown yeast had an increase of oxidative activity in comparison with glucose-grown yeast. In addition to the Qo~ determinations, cytochrome spectra were examined by Biochim. Biophys. Acta, 95 (1965) 6¢o-651
644
C. REILLY, F. SHERMAN
TABLE I RATES
OF
RESPIRATION
(CY P p+) A N D
OF NORMAL
MUTANT
STRAINS
OF YEAST
Y e a s t - s t r a i n s w e r e g r o w n i n 1 % y e a s t e x t r a c t , 2 °/o p e p t o n e a n d v a r i o u s c o n c e n t r a t i o n s (w/v) of d i f f e r e n t c a r b o n sources. T h e r e s p i r a t i o n r a t e s of s t r a i n B-3o6 h a v e b e e n c o r r e c t e d for t h e b a c k m u t a n t s found in some experiments.
Strain number
Dz73-IOB
D332-4A
B-3o6
3789z
Genotype
C Y P p+
cya-5 p+
Plo p+
gltt-x P+
2 ~o dextrose I °/o dextrose 0.25 ~o d e x t r o s e I ~o sucrose I ~o galactose 2 °/o ethanol i % melibiose I °/o raffinose
80 125 12o
o 4° 4°
o o o
3° 25
ioo
i 18 148 I47 134
15
3°
77 61
48
low-temperature spectroscopy. Although the cytochrome content was slightly reduced after growth in YP2 0/oD, as compared to the derepressed condition, all of the cytochromes were clearly visible (Table II). In fact, all of the cytochrome bands, although weak, could be detected in cells that were grown in a medium initially containing 3o % glucose. (Chemical analysis of the glucose concentration at the end of growth showed that the medium still contained IO % glucose.) It is clear that it is difficult to repress completely the synthesis of the cytochromes in normal yeast, even at extremely high glucose concentrations. The normal strain of yeast that was cultivated in raffinose medium had an extremely high concentration of cytochromes, giving the cells a brownish appearance. TABLE II CYTOCHROME CONTENT AND Qo t oF A NORMAL ( D z 7 3 - I o B ) A N D
VARIOUS MUTANT STRAINS
M e a s u r e d a f t e r g r o w t h i n I ~/o y e a s t e x t r a c t , 2 % p e p t o n e a n d e i t h e r 2 ~/o glucose or i ~o melibiose. I n c l u d e d is t h e a b i l i t y of t h e s t r a i n s to u t i l i z e n o n f e r m e n t a b l e c a r b o n s ourc e s for g r o w t h (grow t h on g l y c e r o l m e d i u m ) .
Genotype
C Y P p+ C Y P pcya_ 1 p+
Strain number
D273-IoB D273-IoB-I D234-4D cY3-1 P+ B-254 cY3-~ p+ D3o4-5 B cy3_ 5 p+ D332-4A eYs-s p+ B-4o2 cyl-, eY3-3 p+ D297-4D cYa-x md-cya-1 p+ D313-3C cYa-3/cYa-5 P+ D-345 P,0 P+ B-3o6 gltt_x p+ 37892
Growth on Glucose grown glycerol medium cytoehromes
+ -+ + + ----+ --
-
a
b
c1
c
+
+
+
+ + + ----+
+ + + + + + + +
+ + + + + + + +
+
+
+
+ + ~_ ~ ± ~_ =c 5_i i + +
Qo~ a f t e r g r o w t h on e t h a n o l .
Biochim. Biophys. Acta, 95 (1965) 64o--65I
Melibiose grown Qo~
80 o 62 74 55 o o o o 59 o 3°
cytochromes
Qo~
a
b
c1
c
+
+
+
+ + + + + ~;~ -+ + +
+ + + + + + + +
+ + + + + + + +
+
+
+ + -~z 72 ~_ i i @_ ~ -~ + -~
147 o 86" 98 88 77 58 34 o 94 o 48
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
645
In spite of this, the respiratory activity was consistently slightly less than the melibiose- or ethanol-grown yeast (Table I). It was also noted that the growth rate was markedly decreased in raffinose medium, while only slight differences were observed between growth in glucose and melibiose media (Fig. i; see below). This suggests that the raffinose is so growth-limiting as to give rise to an "unbalanced" synthesis of the cytochrome system. In other words, the catabolism may be so redtlced as to impair the synthesis of a component of the electron transport system, other than a cytochrome.
Growth o/normal and mutants strains o~ yeast in various media During the course of investigating the cytochrome content of mutant strains, it was noted that the growth of certain mutants was more strongly influenced by the type of sugar present in the media. In comparison to normal strains, mutants which were unable to utilize nonfermentable carbon sources for growth, had an extended lag period when cultivated in melibiose or raffinose medium. A complete study of this phenomenon was made by comparing the normal strain (p+) to a vegetative mutant (p-), which was isolated directly trorn the normal strain. Fig. I shows the growth characteristics of p+ and p- strains when grown in media containing either 1 % glLlcose, 1 % melibiose, or 1 % raffinose. It is seen that the p+ strain grows at the same rate and to the same extent in both g]tlcose and melibiose media, although the initial lag phase is slightly prolonged in the latter. I0.0 : )EXTROSE
_
^
5.0 !
I.O
-g
o.5! f
i
1
i
=
l
,
l
i
,
i
r
,
,
i
,
,
i
i
,
,
,
~ 5.o DEXTROSE MELIBIOSE
f.O
0.5:
QI
i
0
i
i
,
,
50
,
i
Hours
I00
,
150
Fig. 1. G r o w t h of t h e n o r m a l y e a s t (top) D273-IOB (CY P p+) a n d of t h e v e g e t a t i v e m u t a n t ( b o t t o m ) D 2 7 3 - I o B - I (CY P p-) in I ~o y e a s t e x t r a c t , 2 ~o p e p t o n e a n d i % of v a r i o u s s u g a r s .
Biochim. Biophys. Acta, 95 (1965) 6 4 o - 6 5 I
0#~
C. REILLY, F. SHERMAN
However, the p- strain has an extended lag phase of about one day in melibiose medium. As previously mentioned, the p+ strain has a delay of about one-half day in raffinose medium and then grows at a reduced rate. In contrast, the p- strain had a marked lag period of over three days in raffinose medium before growth was initiated. When actively growing p- cells were taken from melibiose or raffinose medium and reinoculated into the same types of media, no initial lag period was observed. However, if these cells were first grown for one day in glucose medium, and then inoculated into melibiose or raffinose medium, prolonged lag periods were again seen. It thus appears that there was no selection of cells having increased capacities to utilize melibiose or raffinose, and that the extended lag phase was due to a delay of induced adaptation. Similar differences between p+ and p- strains have been previously noted with growth in melezitose and galactose media 18. It is not known why cytochrome deficiencies increase the delay of synthesis of various inducible enzymes.
Catabolite repression o / c y t o c h r o m e content and respiratory activities in m u t a n t strains
\Vhen a certain class of mutants (cy3_ 5, c y s t , Plo) were incubated on solid YP2 °/oD medium and compared to the same strains cultivated in liquid medium of the same composition, large differences of cytochrome a concentration were noted. This was not observed with normal yeast or other cytochrome-deficient mutants. It appeared that the low concentration of cytochrome a in liquid-grown strains, in comparison to solid-grown strains, could be explained if one assumes that some substance in the medium is limiting at the vicinity of the cells on the agar surface, and that slow diffusion of this substance is equivalent to a lowered concentration. This substance was found to be glucose, since lowering the concentration of glucose in liquid medium resulted in the appearance of cytochrome a. Strains bearing the mutant gene cyl_ 1 have only 5 to IO c~o of the normal concentration of cytochrome c, but have normal or near normal amounts of cytochromes a, b, and c1 (ref. 7). These strains are only slightly deficient in respiration and can grow on most nonfermentable carbon sources. As shown in Table II, the cytochrome pattern of cyl ~ strains was not greatly affected by growth in different types of media. However, cyl_ 1 mutants can be derepressed in a similar manner as wild-type strains. The cy 3 locus is unlinked to cy~, but cy~ mutants are similar to cy 1 mutants by being primarily deficient in cytochrome c (ref. 5). Independent mutants at the cyz locus have been found to have various degrees of deficiencies in cytochrome c, with most mutants having less than 20 °/0 of the normal concentration. cy3_1 and cy 3 ~ strains (two independent mutants of cy3) were similar to normal strains and cy~_ 1 strains inasmuch as their cytochrome patterns and respiratory activities were not greatly influenced by different growth media (Table II). In contrast, cy3_5 strains were very sensitive to catabolite repression. The Qo, of cy3-~ was less than 2 °/o of the Qo~ values of cyz_l or cy3_ ~ after the strains were grown in 2 °/o glucose medium, while there were only slight differences after growth in 1 % melibiose medium (Table III). The respiratory activities of cy3_ 5, which were under Biochim. Biophys, Acta, 95 (1965) 64o-651
047
GLUCOSE REPRESSION OF YEAST CYTOCHROME a TABLE III THE ABILITY OF CYTOCHROME C TO BE REDUCED BY ENDOGENOUS SUBSTRATES
Genotype
C Y P p+ C Y P pPz0 P+ Pz0 P-
Strain number
D273-IOB D273-IOB-I B-3°6 B-3o6- I
Cytochromes
Redox state o[ cytochrome c
a
b
c1
c
+
+
+
+ + + +
i
reduced reduced oxidized reduced
various degrees of catabolite repression, are shown on Table I. The examination of the cytochrome content of cy3_ 5 revealed that the respiratory deficiency, which was observed after growth in 2 ~o glucose medium, was due primarily to the absence of cytochrome a. In other words, melibiose-grown cy3_ 5 had normal or near normal amounts of cytochromes a, b, c1, and was partially deficient in cytochrome c, while glucose-grown c y s _ ~ had almost normal amounts of cytochromes b and Cl, and was partially or completely deficient in cytochrome c, and cytochrome a (Table II). In addition to the cy3_ 5 segregant listed in Table I I (D332-4A) two other independent segregants (D332-2A and D332-IB ) showed a similar glucose sensitivity. The different phenotypes observed among these independent c y 3 mutants cannot be simply explained by the action of modifying genes. A study of a cy3_ 6 strain indicated that its synthesis of cytochrome a was also very sensitive to catabolite repression (Table II). In a few experiments, it appeared as if cY3-s was slightly more sensitive than cy3_5, since there was less cytochrome a and a lower Qo2 in melibiose-grown cyg_ ~ in comparison to melibiose-grown cy~_ 5. The investigation of the haploid recombinant, c y l _ 1 cy3_ 3, further indicated that the increased glucose sensitivity ot cytochrome a synthesis is indeed an indirect effect of the cytochrome c deficiency. While neither c y z _ 1 nor c y s _ 3 shows this effect, c y l _ z cy3_ s was not only sensitive to glucose but appeared to be even more sensitive than cya_6 (Table II). The extent to which cytochrome a synthesis can be blocked is shown with a cy3_~ m d - c y 3 _ l strain, m d - c y 3 _ 1 is a recessive modifying gene, and is unlinked to cy3_ 1 (ref. 5)- Although the nature of the modifying gene is unknown, when it is in combination with Cys_l, cytochrome a is not synthesized, even after the strain is grown in melibiose medium (Table II). Also shown in Table I I are the cytochrome pattern and respiratory activities of the heteroallelic diploid cyz_3/cyg_5, after growth in glucose and melibiose medium. The results clearly indicate that the glucose sensitivity is a recessive property. The important question of whether the increased glucose sensitivity of cytochrome a synthesis is related only to cytochrome c deficiencies, was answered by the examination of Pz0 strains which are completely lacking in, or have only trace amounts of cytochromes b and % This mutant also showed a similar glucose effect, since glucose-grown Pz0 was deficient in cytochromes a, b, and c1, while melibiose-grown Plo was deficient only in cytochromes b, and cz (Table II). However, in contradistinction to cYs-5 and cy3_ e strains, the Pl0 strain was still respiratory deficient after growth in melibiose medium, even though cytochrome a was present. In fact, no respiratory activity (Qo, of less than I) was observed after Plo was grown Biochim. Biophys. Acta, 95 (1965) 64o-65 x
048
c. REILLY, F. SHERMAN
in a number of different media as indicated in Table I. Although some of these experiments were hampered by the presence of backmutants, the Qo, values were equal to zero in the three experiments (growth in 2 % dextrose, I °/o melibiose, and I o~ galactose) in which no backmutants were found. Also when appropriate corrections were made in the remaining experiments, the calculated Qo~ values were also zero. An interesting observation was made confirming the site of the lesion in Pl0Normally, the cytochromes, in a thick suspension of yeast, become quickly reduced and no further reduction is found after the addition of dithionite. This is true not only with normal yeast but with most mutants. However, in a number of experiments with Plo P+, the cytochrome c could not be reduced b y endogenous substrates (Table I I I ) . This effect was more pronounced in cultures having small amounts of cytochrome a. Under these conditions, it was also observed that cytochrome a was slightly oxidized. The inability of endogenous substrates to reduce the cytochromes can be explained b y considering the normal electron transport chain: -->cytochrome b -+ cytochrome q --->cytochrome c -+ cytochrome a -~ 02. Since cytochromes b and c1 are lacking in Pl0, there is little or no transfer of electrons from cytochrome c. Also, cytoehrome c in the double m u t a n t Pz0 P% which is also deficient in cytochrome a, is completely reduced (Table I I I ) . From these experiments with the cytochromedeficient mutants, it is reassuring to note that cytochrome c is oxidized by cytochrome a and reduced b y cytochromes b and c1. In addition to the carbon sources listed in Table II, the following strains were also grown in 1 % raffinose medium: C Y Pp+, C Y P p - , cya_lp +, cy 3 ap +, cY3-5 P+, cY3 6 P+, cYl-1 cya 3 P+, and Pl0 P+. Both the melibiose-grown and raffinosegrown yeast were similar with respect to the presence of cytochrome a. However, as previously mentioned with the normal strain, the m u t a n t strains which were grown in raffinose medium, were higher in cytochrome content, and had slightly lower Qo~ values (approximately 80 %) in comparison to the melibiose-grown strains.
Growth on non/ermentable substrates
An inability to utilize non-fermentable substrates as the sole carbon and energy source is indicative of an ineffective electron-transport system. Shown in Table I I are all strains having either a very marked decrease in growth, or total absence of growth on glycerol medium. Identical results were also obtained with other nonfermentable substrates (ethanol, acetate, pyruvate, DE-lactate, and Llactate). However, some strains showed a slight tendency to grow after prolonged incubation. Small "pinpoint" colonies appeared after one week of incubation of cy3_5 strains. A low proportion (lO-15 %) of cells formed very small colonies after two weeks incubation of cYa-6 and cyl_ 1 cy3_3 strains. A striking correlation exists between the inability to utilize nonfermentable substrates and an increased sensitivity to catabolite repression of cytochrome a synthesis (Table II). All strains which were glucose-sensitive could not utilize nonfermentable substrates (cya_5, cy3_~, cyl_ 1 cy8_3, and Plo) while m a n y of the related mutants had neither of these impairments (cyl_l, cy3_1, and cy~_a). Biochim. Biophys. Acta, 95 (1965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
649
Aconitaseless mutant A glt~_~ mutant was investigated in order to determine the generality that an increased glucose repression of cytochrome a synthesis is associated with mutants having an impaired ability to utilize nonfermentable carbon sources for growth. glt~_1 is a single-gene mutant which is lacking in aconitase and therefore has an impaired respiration with certain substrates, does not grow in media having nonfermentable substrates as a sole carbon source, and has a requirement for glutamate s. The results, which are shown in Tables I and II, clearly show that the concentration of cytochrome a and the respiratory activities in this mutant are not greatly affected by glucose derepression.
Additional pigments in mutant strains In addition to the normal cytochromes, pigments which exhibit absorption bands, have been observed in a number of mutants 6 and in yeast at the end of exponential phase of anaerobic growth 19. cy3_~O+ strains, as well as a number of cytoplasmic (O-) and other genie mutants have a pigment which absorbs at about 575 m/~. The isolation and characterization of this pigment, which is not effected by dithonite (see below), is now in progress in our laboratory. It has been reported ~ that B-3o6 (Plo) has a pigment whose absorption maximum is shifted after treatment with dithionite. However, careful spectroscopic examinations have revealed two broad bands at 575 m# and 584 m#. The addition of dithionite causes the disappearance of the 575 m# band, without effecting the 584 m/~ band. These two pigments are undoubtedly the same as the ones found in anaerobically grown cells TM. Structures for these pigments have been tentatively suggested from their spectroscopic behavior ~9, although isolation and direct identification has not as yet been reported. The concentration of the pigments in Plo have varied considerably from experiment to experiment. Factors controlling their formation have not been fully explored.
DISCUSSION
Glucose and related catabolites are known to repress the synthesis of respiratory enzymes, as well as many other inducible enzymes in a wide variety of organisms. Therefore the present investigation is not only of interest in the understanding of the regulation of cytochrome synthesis, but also in noting any specific interactions which may help to delineate the general mechanism of catabolite (glucose) repression. It is evident that the accentuation of catabolite repression of cytochrome a synthesis can be a secondary effect of cytochrome c deficiencies. The study of cy3 mutants, which are partially deficient in cytochrome c, revealed that mutants (cya_5 and cY3-6) having an ineffective electron transport system (as indicated by an inability to utilize nonfermentable carbon sources) do not synthesize cytochrome a or respire, when grown in a medium containing 2 % glucose. However, growth in Biochim. Biophys. Acta, 95 (I965) 640-651
050
C. REILLY, F. SHERMAN
lower concentrations of glucose, or in melibiose, results in the appearance of cytochrome a, which in some cases (cy3-5) m a y be equal to the normal concentration. Cytochrome a content in other cy3 mutants, which can utilize nonfermentable substrate (cY3_1, cy~_a), is not affected by catabolite repression to any greater degree than normal strains. In this connection it is of importance that cytochrome a synthesis in the double m u t a n t cyl_ 1 cy3_~, which does not utilize nonfermentable substrates, is very sensitive to catabolite repression, while neither of the single mutants, cyl_ 1 and cys_a, have these impairments. A complete block of cytochrome a synthesis was found with the cy3_ Imd-cy3_ 1 mutant. It would be important to determine the types ~ and exact concentrations of cytochrome c in these cy mutants after various degrees of catabolite repression. An increased sensitivity to catabolite repression of cytochrome a synthesis was also observed in a different type of mutant, Pl0, which is primarily deficient in cytochromes b and c1, and which does not respire or utilize nonfermentable carbon sources. This indicates the generality of the phenomenon. The studies with cytochrome-deficient mutants suggest that cytochrome a synthesis is blocked in various degrees depending on the extent of the primary lesion. Cytochrome a synthesis can be considered in three classes of mutants: (I) Cytochrome a synthesis is comparable to normal strains when there is a slight lesion. (2) Cytochrome a synthesis is extremely sensitive to catabolite repression in mutants having a more extensive lesion. (3) No cytochrome a is synthesized in mutants having a "complete" lesion. The conclusion that cytochrome a synthesis is dependent on a functioning cytochrome system can be made b y examining the results from an entirely different investigation 2°. When yeast is cultivated in the presence of antimycin " A " , which specifically blocks electron transport, the synthesis of cytochrome a was inhibited, while there was no effect on cytochromes b and c. At this time it is not known if other lesions, which are not cytochrome deficiencies, can cause cytochrome a synthesis to be more sensitive to catabolite repression. However, the experiments with the aconitaseless mutant, which cannot utilize nonfermentable carbon sources for growth, seem to indicate a direct relationship between the cytochrome system and cytochrome a synthesis. There is a striking parallel between cytochrome-deficient mutants found in yeast and Neurospora crassa. Both " p o k y " (Neurospora) 2x and p- (yeast) ~ strains are deficient in cytochromes a and b, and these deficiencies are inherited cytoplasmically. The single-gene mutants P5 (yeast) ~ and C I I 5 (Neurospora) zx,** lack cytochrome a. The m u t a n t C I I 7 (Neurospora) zl,22, which is deficient in cytochromes a and c, is analogous to mutants described in this investigation, i.e., cY8-5 and cys~ grown in glucose medium. Although no experiments with Neurospora have been reported which indicate a direct relationship between cytochrome content and catabolite repression, an early investigation 2a showed t h a t prolonged cultivation of " p o k y " strains resulted in the appearance of cytochrome oxidase and succinate oxidase activities. Thus it appears that exhaustion of glucose and therefore catabolite derepression, m a y also cause cytochrome synthesis in cytochrome-deficient mutants of Neurospora. I t now seems to be evident that the absence of cytochrome a can be a secondary effect of other cytochrome deficiencies. This m a y account for certain Biochim. Biophys. Acta, 95 (1965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
651
mutants of yeast 4 and Neurospora*l, ~, which have multi-cytochrome deficiencies. Although the exact mechanism is still not known, it can be suggested that the synthesis of cytochrome a is normally regulated by other cytochromes. It can be further suggested that the regulation of enzyme synthesis in a compartmentalized organelle, such as a mitochondrion, may be fundamentally different from other systems which have been extensively investigated 24.
NOTE ADDED IN PROOF
Exact cytochrome c concentrations have been recently determined in a number of cy mutants *~. Cytochrome c content was considerably lower in all mutants in which cytochrome a synthesis was very sensitive to catabolite repression, and which did not utilize non-fermentable carbon sources. ACKNOWLEDGEMENTS
This investigation was supported by the U. S. Atomic Energy Commission at The University of Rochester Atomic Energy Project, Rochester, New York. Dr. C. REILLY was a visiting Fulbright Scholar. REFERENCES
I I. SEKUZU, H. MIZUSHIMA AND K. OKUNUKI, Biochim. Biophys. Acta, 85 (1964) 516. 2 F. SHERMAN, Genetics, 48 (1963) 375. 3 B. EPHRIJSSI, Nucleo-cytoplasmie Relations in Microorganisms, C l a r e n d o n Press, Oxford, 1953, p. 13. 4 F. SHERMAN AND P. P. SLONIMSKI, Biochim. Biophys. Acta, 90 (1964) I. 5 V. SHERMAN,Genetics, 49 (1964) 39. 6 F. SHERMAN, Intern. Syrup. on Mechanisms o~ Regulation o/ Cellular Activities in Microorganisms, Marseille, 1963, G o r d o n a n d Breach, N e w York, 1964. 7 P. P. SLONIMSKI AND F. SHERMAN,in p r e p a r a t i o n . 8 M. OGUR, L. COKER AND S. OGUR, Biochem. Biophys. Res. Commun., 14 (1964) 193. 9 M. OGUR, R. ST. JOHN AND S. NAGAI, Science, 125 (1957) 928. IO F. SHERMAN AND ]3. EPHRUSSI, Genetics, 47 (1962) 695. I I D. PITTMAN, J. M. WEBB, A. ROSHANMANESH AND L. E. COKER, Genetics, 45 (196o) lO23I2 B. EPHRUSSI, P. P. SLONIMSKI, Y. YOTSUYANAGI AND J. TAVLITZKI, Compt. Rend. Tray. Lab. Carlsberg, Sdr. Physiol., 26 (1956) 87. 13 C. F. STRITTMATTER,J. Gen. Microbiol., 16 (1957) 169. 14 P. P. SLONIMSKI, Soc. Chem. Ind. London Monograph, 3 (1958) 7. 15 Y. YOTSUYANAGI, J. Ultrastruct. Res., 7 (1962) 121. I6 G. SCHATZ,Biochem. Biophys. Res. Commun., I2 (1963) 448. 17 E. S. POLAKIS, W. BARTLE¥ AND G. A. MEEK, Biochem. J . , 90 (1964) 369. 18 C. C. LINDEGREN, M. OGUR, I). PITTMAN AND G. LINDEGREN, Science, 126 (1957) 398. 19 P. CHAIX AND P. LABBE, Intern. Syrup. on Mechanisms o/ Regulation o~ Cellular Activities in Microorganisms, Marseille, 1963, G o r d o n a n d Breach, N e w York, 1964. 20 M. Y~AS, Exptl. Cell Res., i i (1956) I. 21 M. B. MITCHELL, H. K. MITCHELL AND A. TISSI]~RES, Proc. Natl. Acad. Sci. U.S., 39 (1953) 606. 22 A. TlSSI¢~RES AND H. K. MITCHELL, J. Biol. Chem., 2o8 (1954) 241. 23 F. A. HASKINS, A. TlSSI~RES, H. I~. MITCHELL AND M. B. MITCHELL, J. Biol. Chem., 200 (1953) 819. 24 F o r e x a m p l e , v a r i o u s p a p e r s in Cold Spring Harbor Syrup. (2uant. Biol., 26 (1961). 25 F. SHERMAN, H. TABER AND W . CAMPBELL, i n p r e p a r a t i o n .
Received January Ilth, 196 5 Biochim. Biophys. Acta, 95 (1965) 64o-651
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95165
GLUCOSE R E P R E S S I O N OF CYTOCHROME a S Y N T H E S I S IN C Y T O C H R O M E - D E F I C I E N T MUTANTS OF YEAST
CONOR REILLY ANDFRED SHERMAN Department o[ Radiation Biology, University o/Rochester School o[ Medicine and Dentistry, Rochester, N.Y. (U.S.A.) (Received September 2rid, 1964)
SUMMARY I. Cytochrome content and respiratory activities were investigated in normal and cytochrome-deficient mutants of yeast which were under various degrees of catabolite (glucose) repression. 2. Derepression could be obtained b y growing yeast in melibiose, probably due to the low rate of utilization of this felmentable sugar. 3. The synthesis of cytochrome a (i.e., cytochromes a and aa) was very sensitive to catabolite repression in single-gene mutants (cya_ 5 and cy3_~) and a doublegene m u t a n t (cyl_lcys_3), which were partially deficient in cytochrome c, and in a single-gene m u t a n t (Pl0), which was deficient in cytochromes b and c1. Cytochrome a synthesis behaved normally in other cytochrome c deficient mutants (cYl-1, cy~-l, and cy34 ). In some strains, cytochrome a synthesis was completely blocked. 4- I t is suggested that the synthesis of cytochrome a is normally regulated by other cytochromes.
INTRODUCTION Normal, aerobically-grown Saccharomyces cerevisiae has a classical cytochrome system, containing cytochromes a, b, cl, and c. In spite of the fact that these cytochromes are distinct and separable proteins 1, certain single-gene mutants ~ and cytoplasmic mutants 3 have been found to have multiple deficiencies 4. Although a number of tentative theories have been presented 4, no clear explanation has been given to account for a single gene controlling more than one cytochrome. A large number of cytochrome-deficient mutants have been iecently isolated b y a new procedure 5. The present investigation of the cytochrome content of some of these mutants has revealed the dependence of the synthesis of cytochrome a (i.e., cytochromes a and c~) on the presence of other cytochromes. The synthesis of Abbreviations used for culture media: Y, I ~O Bacto-yeast extract; P, 2 °/o Bactopeptone; D, dextrose (with percentage indicated); M, melibiose (with percentage indicated); G, glycerol (with percentage indicated). Biochim. Biophys. Acta, 95 (1965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
641
cytochrome a, in certain cytochrome c-deficient mutants and cytochrome bdeficient mutants, has been found to be extremely sensitive to glucose repression.
MATERIALS AND
METHODS
Nomenclature
The original symbols~, 6 used to designate m u t a n t characters controlling the cytochrome and related systems are: p, for single-gene mutants which are unable to grow on nonfermentable substrates (e.g. glycerol, lactate, ethanol and acetate) as the sole carbon source, and which m a y or m a y not have an altered absorption spectrum; cy, for single-gene mutants having an altered absolption spectrum (e.g., the loss of a cytochrome) but still retaining the ability to utilize nonfermentable carbon sources; and p - f)r cytJplasmic mutants a which are unable to utilize nonfermentable carbon sources. The symbols employed to desiglaate these wild type characters are: P C Y p+. However, the terminology is somewhat misleading since, not only is an artificial distinction made between cy and p mutants, but independent mutants at the same locus can have either a p or cy phenotype. For example, cy3_1, cya-3, cY~-5, and cyg_6 are all mutants at the same locus, but cy3_ ~ and cyg-n can utilize nonfermentable carbon somces, while cy3-5 and cy3-8 cannot. Also m a n y cy mutants have a pronounced decrease of growth rates in synthetic media containing certain nonfermentable substrates as the sole carbon source We will, however, retain the original symbols as previously published.
Strains and genetics
The origin and genetics of the m u t a n t genes cyl_ 1 (ref. 7), cy3-1, cy~-3, and md-cyg_ ~ (ref. 5) have been described. B-3o6 (Pa0) was induced b y nitrous acid and detected b y the spectroscopic method s. In a cross to a normal strain, Pl0 segregated as a single-gene in five asci. The method of detecting cy3_5 and cy3_~, as well as other cytochrome-deficient mutants, will be described elsewhere, gltl_ 1 (strain number 37892) was kindly given to us by Dr. M. OGURs.
Culture methods
Liquid growth media contained 1 % Bacto-yeast extract, 2 % Bacto-peptone, and various concentrations of different carbon sources which are indicated as follows: Y P 2 % D , 2 % dextrose; Y P I % D , 1 % dextrose; Y P I % M , 1 % melibiose, etc. Two percent agar was added in preparing solid media. Freshly grown cells were inoculated to lO 6 cells/ml and incubated at 3 °0 with vigorous shaking. In order to assure good aeration, the erlenmeyer growth flasks were never filled to more than IO % of their total volume. Unless stated otherwise, cells were harvested at early stationary phase of growth. 13iochim. Biophys. Acta, 95 (1965) 640-651
042
C. REILLY, F. SHERMAN
Growth rates were determined with 125 ml Nephelo-culture flasks containing io ml of media. The absorbancy of cultures was measured at 45 ° m/z with a Spectronic 20 spectrophotometer (Bausch and Lomb). Special reference standards were employed in order to measure the entire growth curve without diluting the culture at high densities.
Respiration Respiration rates of intact cells were determined by the direct Warburg method at 3 °° in the presence of air. 2,0 ml of reactive material contained: o.i M glucose; 67 mM KH2PO 4 at p H 4.6; and 2.5 mg (dry wt) of yeast. 0.2 ml of 20 % KOH was added to the center well and sometimes to the side arm when utilizing strains having very low rates of oxygen consumFtion. Qo2 was expressed as #1 of oxygen consumed per mg dry wt. of yeast per hour.
Cytochrome spectra The cytochrome content of intact cells was estimated by low-temperature (--19 o°) spectroscopy by the method previously described by SHERMAN AND SLONIMSKI4. In some instances, low-temperature recordings were also made 6.
Homogeneity of cell populations Obviously, relatively homogeneous cell suspensions should be used for measurements of respiration and cytochrome content. The frequencies of backmutants in mutant strains, which are unable to grow on nonfermentable carbon sources, can be assayed by simply plating the cell suspension and determining the proportion of cells giving rise to colonies on YP3 %G (I % yeast extract, 2 % peptone, and 3 % glycerol). In all strains employed in the present study, the proportion of backmutants was negligible except for strain B-3o6 (Pl0), which had from o to 25 % in various experiments. In these cases corrections have been made as indicated below. In the study of cytochrome mutants, one possible source of error is the relatively high frequencies of p p- cells found in some p p+ cultures ~. Although the frequency of p- cells can be easily determined in normal strains by the triphenyltetrazolium chloride-overlay method 9, or by plating on YP o. I°/oD3°/o G (I % yeast extract, 2 % peptone, o.I °/o dextrose, and 3 % glycerol), it is more difficult to' make such determinations with certain mutant strains. Two methods have been employed for determining the exact frequencies of p- cells in mutant cultures: (I) When zygotes from a cross of the mutant with a neutral vegetative mutant are plated, only the p p- cells form p- hybrids 1°. (2) Sometimes the pigment produced by adI (adenine dependence) mutants is affected by the cytochrome phenotype so that p p+ and p pcolonies exhibit distinct colors1°, 11. In the investigation presented herein, we have developed still another method which has certain obvious advantages. Approx. 5O-lOO cells, from the haploid population to be tested, were plated Biochim. Biophys. Acta, 95 (I965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
643
per YP2~oD plate and the plates incubated for about three days until the colonies were formed. A second plate was then prepared by streaking approx, lO8 cells per plate of a P p- strain of the opposite mating type. The first plate, which contained the colonies to be tested, was replicaplated on the second plate, and the mating mixture incubated for one day. This plate was either stained directly with triphenyltetrazolium chloride, or replicaplated on Y P 3 % G . In both cases, the frequency of p- ceils in the original population can be inferred from the frequency of p- hybrid colonies. The frequencies of p- cells in all normal and mutant strains were determined either directly by plating on YP o. I ~o D3 ~o G (in cases when the strain in question could utilize nonfermentable carbon sources) or indirectly by the mating method described above. In all cases, the frequencies of p- cells were negligible, constituting less than IO % of the cell populations.
RESULTS
Characteristics o[ normal yeast grown with various carbon sources
Aerobic growth of normal strains of yeast in high concentrations of glucose results in lowering of the Qov cytochromes and related enzymes, and mitochondrial formation 12-17. It is well known that glucose repression of the cytochrome system can be alleviated by growth in nonfermentable carbon sources. However, as previously pointed out ~, this method cannot be employed to derepress certain mutants which are unable to utilize nonfermentable substrates for growth. It is possible to circumvent this condition by growing the mutants in limiting glucose, as in a chemostat, or by feeding a limiting amount of glucose to the growing culture. Since these procedures require special equipment and are cumbersome for the study of large numbers of strains, we have investigated conditions which simulate low concentrations of glucose, and still produce a high yield of cells. It was reasoned that the catabolism of certain fermentable substrates could be limiting, due to low concentrations of hydrolyzing or other enzymes. With this goal in mind, a normal yeast strain (D273loB) was grown in a number of media containing various fermentable sugars, and a nonfermentable carbon source (ethanol). As shown in Table I, the completely derepressed strain, which was grown in ethanol, had a Qo2 of approx. 15o. The same rate of respiration could be obtained after growth in melibiose. No decrease in catabolite (glucose) repression was observed after growth in sucrose or galactose (Table I). Other carbohydrates (mannose, trehalose, melezitose, and starch) either could not be utilized by the yeast, or also failed to diminish catabolite repression. As will be discussed below, an increase of oxidative activity was also observed after growth in raffinose (Table I). It was therefore concluded that melibiose is the best fermentable sugar to use for obtaining derepressed yeast. In retrospect, the same reasoning can explain the early observation of STRITTMATTER13 that a specific galactose-grown yeast had an increase of oxidative activity in comparison with glucose-grown yeast. In addition to the Qo~ determinations, cytochrome spectra were examined by Biochim. Biophys. Acta, 95 (1965) 6¢o-651
644
C. REILLY, F. SHERMAN
TABLE I RATES
OF
RESPIRATION
(CY P p+) A N D
OF NORMAL
MUTANT
STRAINS
OF YEAST
Y e a s t - s t r a i n s w e r e g r o w n i n 1 % y e a s t e x t r a c t , 2 °/o p e p t o n e a n d v a r i o u s c o n c e n t r a t i o n s (w/v) of d i f f e r e n t c a r b o n sources. T h e r e s p i r a t i o n r a t e s of s t r a i n B-3o6 h a v e b e e n c o r r e c t e d for t h e b a c k m u t a n t s found in some experiments.
Strain number
Dz73-IOB
D332-4A
B-3o6
3789z
Genotype
C Y P p+
cya-5 p+
Plo p+
gltt-x P+
2 ~o dextrose I °/o dextrose 0.25 ~o d e x t r o s e I ~o sucrose I ~o galactose 2 °/o ethanol i % melibiose I °/o raffinose
80 125 12o
o 4° 4°
o o o
3° 25
ioo
i 18 148 I47 134
15
3°
77 61
48
low-temperature spectroscopy. Although the cytochrome content was slightly reduced after growth in YP2 0/oD, as compared to the derepressed condition, all of the cytochromes were clearly visible (Table II). In fact, all of the cytochrome bands, although weak, could be detected in cells that were grown in a medium initially containing 3o % glucose. (Chemical analysis of the glucose concentration at the end of growth showed that the medium still contained IO % glucose.) It is clear that it is difficult to repress completely the synthesis of the cytochromes in normal yeast, even at extremely high glucose concentrations. The normal strain of yeast that was cultivated in raffinose medium had an extremely high concentration of cytochromes, giving the cells a brownish appearance. TABLE II CYTOCHROME CONTENT AND Qo t oF A NORMAL ( D z 7 3 - I o B ) A N D
VARIOUS MUTANT STRAINS
M e a s u r e d a f t e r g r o w t h i n I ~/o y e a s t e x t r a c t , 2 % p e p t o n e a n d e i t h e r 2 ~/o glucose or i ~o melibiose. I n c l u d e d is t h e a b i l i t y of t h e s t r a i n s to u t i l i z e n o n f e r m e n t a b l e c a r b o n s ourc e s for g r o w t h (grow t h on g l y c e r o l m e d i u m ) .
Genotype
C Y P p+ C Y P pcya_ 1 p+
Strain number
D273-IoB D273-IoB-I D234-4D cY3-1 P+ B-254 cY3-~ p+ D3o4-5 B cy3_ 5 p+ D332-4A eYs-s p+ B-4o2 cyl-, eY3-3 p+ D297-4D cYa-x md-cya-1 p+ D313-3C cYa-3/cYa-5 P+ D-345 P,0 P+ B-3o6 gltt_x p+ 37892
Growth on Glucose grown glycerol medium cytoehromes
+ -+ + + ----+ --
-
a
b
c1
c
+
+
+
+ + + ----+
+ + + + + + + +
+ + + + + + + +
+
+
+
+ + ~_ ~ ± ~_ =c 5_i i + +
Qo~ a f t e r g r o w t h on e t h a n o l .
Biochim. Biophys. Acta, 95 (1965) 64o--65I
Melibiose grown Qo~
80 o 62 74 55 o o o o 59 o 3°
cytochromes
Qo~
a
b
c1
c
+
+
+
+ + + + + ~;~ -+ + +
+ + + + + + + +
+ + + + + + + +
+
+
+ + -~z 72 ~_ i i @_ ~ -~ + -~
147 o 86" 98 88 77 58 34 o 94 o 48
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
645
In spite of this, the respiratory activity was consistently slightly less than the melibiose- or ethanol-grown yeast (Table I). It was also noted that the growth rate was markedly decreased in raffinose medium, while only slight differences were observed between growth in glucose and melibiose media (Fig. i; see below). This suggests that the raffinose is so growth-limiting as to give rise to an "unbalanced" synthesis of the cytochrome system. In other words, the catabolism may be so redtlced as to impair the synthesis of a component of the electron transport system, other than a cytochrome.
Growth o/normal and mutants strains o~ yeast in various media During the course of investigating the cytochrome content of mutant strains, it was noted that the growth of certain mutants was more strongly influenced by the type of sugar present in the media. In comparison to normal strains, mutants which were unable to utilize nonfermentable carbon sources for growth, had an extended lag period when cultivated in melibiose or raffinose medium. A complete study of this phenomenon was made by comparing the normal strain (p+) to a vegetative mutant (p-), which was isolated directly trorn the normal strain. Fig. I shows the growth characteristics of p+ and p- strains when grown in media containing either 1 % glLlcose, 1 % melibiose, or 1 % raffinose. It is seen that the p+ strain grows at the same rate and to the same extent in both g]tlcose and melibiose media, although the initial lag phase is slightly prolonged in the latter. I0.0 : )EXTROSE
_
^
5.0 !
I.O
-g
o.5! f
i
1
i
=
l
,
l
i
,
i
r
,
,
i
,
,
i
i
,
,
,
~ 5.o DEXTROSE MELIBIOSE
f.O
0.5:
QI
i
0
i
i
,
,
50
,
i
Hours
I00
,
150
Fig. 1. G r o w t h of t h e n o r m a l y e a s t (top) D273-IOB (CY P p+) a n d of t h e v e g e t a t i v e m u t a n t ( b o t t o m ) D 2 7 3 - I o B - I (CY P p-) in I ~o y e a s t e x t r a c t , 2 ~o p e p t o n e a n d i % of v a r i o u s s u g a r s .
Biochim. Biophys. Acta, 95 (1965) 6 4 o - 6 5 I
0#~
C. REILLY, F. SHERMAN
However, the p- strain has an extended lag phase of about one day in melibiose medium. As previously mentioned, the p+ strain has a delay of about one-half day in raffinose medium and then grows at a reduced rate. In contrast, the p- strain had a marked lag period of over three days in raffinose medium before growth was initiated. When actively growing p- cells were taken from melibiose or raffinose medium and reinoculated into the same types of media, no initial lag period was observed. However, if these cells were first grown for one day in glucose medium, and then inoculated into melibiose or raffinose medium, prolonged lag periods were again seen. It thus appears that there was no selection of cells having increased capacities to utilize melibiose or raffinose, and that the extended lag phase was due to a delay of induced adaptation. Similar differences between p+ and p- strains have been previously noted with growth in melezitose and galactose media 18. It is not known why cytochrome deficiencies increase the delay of synthesis of various inducible enzymes.
Catabolite repression o / c y t o c h r o m e content and respiratory activities in m u t a n t strains
\Vhen a certain class of mutants (cy3_ 5, c y s t , Plo) were incubated on solid YP2 °/oD medium and compared to the same strains cultivated in liquid medium of the same composition, large differences of cytochrome a concentration were noted. This was not observed with normal yeast or other cytochrome-deficient mutants. It appeared that the low concentration of cytochrome a in liquid-grown strains, in comparison to solid-grown strains, could be explained if one assumes that some substance in the medium is limiting at the vicinity of the cells on the agar surface, and that slow diffusion of this substance is equivalent to a lowered concentration. This substance was found to be glucose, since lowering the concentration of glucose in liquid medium resulted in the appearance of cytochrome a. Strains bearing the mutant gene cyl_ 1 have only 5 to IO c~o of the normal concentration of cytochrome c, but have normal or near normal amounts of cytochromes a, b, and c1 (ref. 7). These strains are only slightly deficient in respiration and can grow on most nonfermentable carbon sources. As shown in Table II, the cytochrome pattern of cyl ~ strains was not greatly affected by growth in different types of media. However, cyl_ 1 mutants can be derepressed in a similar manner as wild-type strains. The cy 3 locus is unlinked to cy~, but cy~ mutants are similar to cy 1 mutants by being primarily deficient in cytochrome c (ref. 5). Independent mutants at the cyz locus have been found to have various degrees of deficiencies in cytochrome c, with most mutants having less than 20 °/0 of the normal concentration. cy3_1 and cy 3 ~ strains (two independent mutants of cy3) were similar to normal strains and cy~_ 1 strains inasmuch as their cytochrome patterns and respiratory activities were not greatly influenced by different growth media (Table II). In contrast, cy3_5 strains were very sensitive to catabolite repression. The Qo, of cy3-~ was less than 2 °/o of the Qo~ values of cyz_l or cy3_ ~ after the strains were grown in 2 °/o glucose medium, while there were only slight differences after growth in 1 % melibiose medium (Table III). The respiratory activities of cy3_ 5, which were under Biochim. Biophys, Acta, 95 (1965) 64o-651
047
GLUCOSE REPRESSION OF YEAST CYTOCHROME a TABLE III THE ABILITY OF CYTOCHROME C TO BE REDUCED BY ENDOGENOUS SUBSTRATES
Genotype
C Y P p+ C Y P pPz0 P+ Pz0 P-
Strain number
D273-IOB D273-IOB-I B-3°6 B-3o6- I
Cytochromes
Redox state o[ cytochrome c
a
b
c1
c
+
+
+
+ + + +
i
reduced reduced oxidized reduced
various degrees of catabolite repression, are shown on Table I. The examination of the cytochrome content of cy3_ 5 revealed that the respiratory deficiency, which was observed after growth in 2 ~o glucose medium, was due primarily to the absence of cytochrome a. In other words, melibiose-grown cy3_ 5 had normal or near normal amounts of cytochromes a, b, c1, and was partially deficient in cytochrome c, while glucose-grown c y s _ ~ had almost normal amounts of cytochromes b and Cl, and was partially or completely deficient in cytochrome c, and cytochrome a (Table II). In addition to the cy3_ 5 segregant listed in Table I I (D332-4A) two other independent segregants (D332-2A and D332-IB ) showed a similar glucose sensitivity. The different phenotypes observed among these independent c y 3 mutants cannot be simply explained by the action of modifying genes. A study of a cy3_ 6 strain indicated that its synthesis of cytochrome a was also very sensitive to catabolite repression (Table II). In a few experiments, it appeared as if cY3-s was slightly more sensitive than cy3_5, since there was less cytochrome a and a lower Qo2 in melibiose-grown cyg_ ~ in comparison to melibiose-grown cy~_ 5. The investigation of the haploid recombinant, c y l _ 1 cy3_ 3, further indicated that the increased glucose sensitivity ot cytochrome a synthesis is indeed an indirect effect of the cytochrome c deficiency. While neither c y z _ 1 nor c y s _ 3 shows this effect, c y l _ z cy3_ s was not only sensitive to glucose but appeared to be even more sensitive than cya_6 (Table II). The extent to which cytochrome a synthesis can be blocked is shown with a cy3_~ m d - c y 3 _ l strain, m d - c y 3 _ 1 is a recessive modifying gene, and is unlinked to cy3_ 1 (ref. 5)- Although the nature of the modifying gene is unknown, when it is in combination with Cys_l, cytochrome a is not synthesized, even after the strain is grown in melibiose medium (Table II). Also shown in Table I I are the cytochrome pattern and respiratory activities of the heteroallelic diploid cyz_3/cyg_5, after growth in glucose and melibiose medium. The results clearly indicate that the glucose sensitivity is a recessive property. The important question of whether the increased glucose sensitivity of cytochrome a synthesis is related only to cytochrome c deficiencies, was answered by the examination of Pz0 strains which are completely lacking in, or have only trace amounts of cytochromes b and % This mutant also showed a similar glucose effect, since glucose-grown Pz0 was deficient in cytochromes a, b, and c1, while melibiose-grown Plo was deficient only in cytochromes b, and cz (Table II). However, in contradistinction to cYs-5 and cy3_ e strains, the Pl0 strain was still respiratory deficient after growth in melibiose medium, even though cytochrome a was present. In fact, no respiratory activity (Qo, of less than I) was observed after Plo was grown Biochim. Biophys. Acta, 95 (1965) 64o-65 x
048
c. REILLY, F. SHERMAN
in a number of different media as indicated in Table I. Although some of these experiments were hampered by the presence of backmutants, the Qo, values were equal to zero in the three experiments (growth in 2 % dextrose, I °/o melibiose, and I o~ galactose) in which no backmutants were found. Also when appropriate corrections were made in the remaining experiments, the calculated Qo~ values were also zero. An interesting observation was made confirming the site of the lesion in Pl0Normally, the cytochromes, in a thick suspension of yeast, become quickly reduced and no further reduction is found after the addition of dithionite. This is true not only with normal yeast but with most mutants. However, in a number of experiments with Plo P+, the cytochrome c could not be reduced b y endogenous substrates (Table I I I ) . This effect was more pronounced in cultures having small amounts of cytochrome a. Under these conditions, it was also observed that cytochrome a was slightly oxidized. The inability of endogenous substrates to reduce the cytochromes can be explained b y considering the normal electron transport chain: -->cytochrome b -+ cytochrome q --->cytochrome c -+ cytochrome a -~ 02. Since cytochromes b and c1 are lacking in Pl0, there is little or no transfer of electrons from cytochrome c. Also, cytoehrome c in the double m u t a n t Pz0 P% which is also deficient in cytochrome a, is completely reduced (Table I I I ) . From these experiments with the cytochromedeficient mutants, it is reassuring to note that cytochrome c is oxidized by cytochrome a and reduced b y cytochromes b and c1. In addition to the carbon sources listed in Table II, the following strains were also grown in 1 % raffinose medium: C Y Pp+, C Y P p - , cya_lp +, cy 3 ap +, cY3-5 P+, cY3 6 P+, cYl-1 cya 3 P+, and Pl0 P+. Both the melibiose-grown and raffinosegrown yeast were similar with respect to the presence of cytochrome a. However, as previously mentioned with the normal strain, the m u t a n t strains which were grown in raffinose medium, were higher in cytochrome content, and had slightly lower Qo~ values (approximately 80 %) in comparison to the melibiose-grown strains.
Growth on non/ermentable substrates
An inability to utilize non-fermentable substrates as the sole carbon and energy source is indicative of an ineffective electron-transport system. Shown in Table I I are all strains having either a very marked decrease in growth, or total absence of growth on glycerol medium. Identical results were also obtained with other nonfermentable substrates (ethanol, acetate, pyruvate, DE-lactate, and Llactate). However, some strains showed a slight tendency to grow after prolonged incubation. Small "pinpoint" colonies appeared after one week of incubation of cy3_5 strains. A low proportion (lO-15 %) of cells formed very small colonies after two weeks incubation of cYa-6 and cyl_ 1 cy3_3 strains. A striking correlation exists between the inability to utilize nonfermentable substrates and an increased sensitivity to catabolite repression of cytochrome a synthesis (Table II). All strains which were glucose-sensitive could not utilize nonfermentable substrates (cya_5, cy3_~, cyl_ 1 cy8_3, and Plo) while m a n y of the related mutants had neither of these impairments (cyl_l, cy3_1, and cy~_a). Biochim. Biophys. Acta, 95 (1965) 64o-651
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Aconitaseless mutant A glt~_~ mutant was investigated in order to determine the generality that an increased glucose repression of cytochrome a synthesis is associated with mutants having an impaired ability to utilize nonfermentable carbon sources for growth. glt~_1 is a single-gene mutant which is lacking in aconitase and therefore has an impaired respiration with certain substrates, does not grow in media having nonfermentable substrates as a sole carbon source, and has a requirement for glutamate s. The results, which are shown in Tables I and II, clearly show that the concentration of cytochrome a and the respiratory activities in this mutant are not greatly affected by glucose derepression.
Additional pigments in mutant strains In addition to the normal cytochromes, pigments which exhibit absorption bands, have been observed in a number of mutants 6 and in yeast at the end of exponential phase of anaerobic growth 19. cy3_~O+ strains, as well as a number of cytoplasmic (O-) and other genie mutants have a pigment which absorbs at about 575 m/~. The isolation and characterization of this pigment, which is not effected by dithonite (see below), is now in progress in our laboratory. It has been reported ~ that B-3o6 (Plo) has a pigment whose absorption maximum is shifted after treatment with dithionite. However, careful spectroscopic examinations have revealed two broad bands at 575 m# and 584 m#. The addition of dithionite causes the disappearance of the 575 m# band, without effecting the 584 m/~ band. These two pigments are undoubtedly the same as the ones found in anaerobically grown cells TM. Structures for these pigments have been tentatively suggested from their spectroscopic behavior ~9, although isolation and direct identification has not as yet been reported. The concentration of the pigments in Plo have varied considerably from experiment to experiment. Factors controlling their formation have not been fully explored.
DISCUSSION
Glucose and related catabolites are known to repress the synthesis of respiratory enzymes, as well as many other inducible enzymes in a wide variety of organisms. Therefore the present investigation is not only of interest in the understanding of the regulation of cytochrome synthesis, but also in noting any specific interactions which may help to delineate the general mechanism of catabolite (glucose) repression. It is evident that the accentuation of catabolite repression of cytochrome a synthesis can be a secondary effect of cytochrome c deficiencies. The study of cy3 mutants, which are partially deficient in cytochrome c, revealed that mutants (cya_5 and cY3-6) having an ineffective electron transport system (as indicated by an inability to utilize nonfermentable carbon sources) do not synthesize cytochrome a or respire, when grown in a medium containing 2 % glucose. However, growth in Biochim. Biophys. Acta, 95 (I965) 640-651
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C. REILLY, F. SHERMAN
lower concentrations of glucose, or in melibiose, results in the appearance of cytochrome a, which in some cases (cy3-5) m a y be equal to the normal concentration. Cytochrome a content in other cy3 mutants, which can utilize nonfermentable substrate (cY3_1, cy~_a), is not affected by catabolite repression to any greater degree than normal strains. In this connection it is of importance that cytochrome a synthesis in the double m u t a n t cyl_ 1 cy3_~, which does not utilize nonfermentable substrates, is very sensitive to catabolite repression, while neither of the single mutants, cyl_ 1 and cys_a, have these impairments. A complete block of cytochrome a synthesis was found with the cy3_ Imd-cy3_ 1 mutant. It would be important to determine the types ~ and exact concentrations of cytochrome c in these cy mutants after various degrees of catabolite repression. An increased sensitivity to catabolite repression of cytochrome a synthesis was also observed in a different type of mutant, Pl0, which is primarily deficient in cytochromes b and c1, and which does not respire or utilize nonfermentable carbon sources. This indicates the generality of the phenomenon. The studies with cytochrome-deficient mutants suggest that cytochrome a synthesis is blocked in various degrees depending on the extent of the primary lesion. Cytochrome a synthesis can be considered in three classes of mutants: (I) Cytochrome a synthesis is comparable to normal strains when there is a slight lesion. (2) Cytochrome a synthesis is extremely sensitive to catabolite repression in mutants having a more extensive lesion. (3) No cytochrome a is synthesized in mutants having a "complete" lesion. The conclusion that cytochrome a synthesis is dependent on a functioning cytochrome system can be made b y examining the results from an entirely different investigation 2°. When yeast is cultivated in the presence of antimycin " A " , which specifically blocks electron transport, the synthesis of cytochrome a was inhibited, while there was no effect on cytochromes b and c. At this time it is not known if other lesions, which are not cytochrome deficiencies, can cause cytochrome a synthesis to be more sensitive to catabolite repression. However, the experiments with the aconitaseless mutant, which cannot utilize nonfermentable carbon sources for growth, seem to indicate a direct relationship between the cytochrome system and cytochrome a synthesis. There is a striking parallel between cytochrome-deficient mutants found in yeast and Neurospora crassa. Both " p o k y " (Neurospora) 2x and p- (yeast) ~ strains are deficient in cytochromes a and b, and these deficiencies are inherited cytoplasmically. The single-gene mutants P5 (yeast) ~ and C I I 5 (Neurospora) zx,** lack cytochrome a. The m u t a n t C I I 7 (Neurospora) zl,22, which is deficient in cytochromes a and c, is analogous to mutants described in this investigation, i.e., cY8-5 and cys~ grown in glucose medium. Although no experiments with Neurospora have been reported which indicate a direct relationship between cytochrome content and catabolite repression, an early investigation 2a showed t h a t prolonged cultivation of " p o k y " strains resulted in the appearance of cytochrome oxidase and succinate oxidase activities. Thus it appears that exhaustion of glucose and therefore catabolite derepression, m a y also cause cytochrome synthesis in cytochrome-deficient mutants of Neurospora. I t now seems to be evident that the absence of cytochrome a can be a secondary effect of other cytochrome deficiencies. This m a y account for certain Biochim. Biophys. Acta, 95 (1965) 64o-651
GLUCOSE REPRESSION OF YEAST CYTOCHROME a
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mutants of yeast 4 and Neurospora*l, ~, which have multi-cytochrome deficiencies. Although the exact mechanism is still not known, it can be suggested that the synthesis of cytochrome a is normally regulated by other cytochromes. It can be further suggested that the regulation of enzyme synthesis in a compartmentalized organelle, such as a mitochondrion, may be fundamentally different from other systems which have been extensively investigated 24.
NOTE ADDED IN PROOF
Exact cytochrome c concentrations have been recently determined in a number of cy mutants *~. Cytochrome c content was considerably lower in all mutants in which cytochrome a synthesis was very sensitive to catabolite repression, and which did not utilize non-fermentable carbon sources. ACKNOWLEDGEMENTS
This investigation was supported by the U. S. Atomic Energy Commission at The University of Rochester Atomic Energy Project, Rochester, New York. Dr. C. REILLY was a visiting Fulbright Scholar. REFERENCES
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Received January Ilth, 196 5 Biochim. Biophys. Acta, 95 (1965) 64o-651