Derepression of mitochondria and their enzymes in yeast: Regulatory aspects

ARCHIVES

OF

BIOCHEMISTRY

Derepression

AND

BIOPHYSICS

162, 248-271

of Mitochondria

and

Regulatory PHILIP Department

S. PERLMAN

(1974)

their

Enzymes

in Yeast:

Aspects’ HENRY

AND

R. MAHLER2

of Genetics, Ohio State University, Columbus, Ohio 4.9210and Department of Chemistry, Indiana University,3 Rloomington, Indiana 47401 Received

November

12, 1973

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate: cytochrome c reductase and NADH: cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for n-malate, L-glutamate, and L,-isocitrate. Cytochromes b, cl, and aa are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected. We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4oj,; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa3, b, and cl; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than g-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression. The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.

The occurrence of catabolite repression in and related species (glucose effect, inverse Pasteur effect) was first described exSaccharmnyces cerevisiae (e.g., S. carlsbergensis) 1 Supported The National

by Research Grant GM 12228 from Institute of General Medical Sci-

ences

Healt;l National Service Institutes

* Recipient of Public Health Career Award GM-05060 from General Medical Sciences. 3 Publication No. 2357.

248 Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

Inc. reserved.

of Health,

U.S. Public

Service Research the Institute of

l)licitly by Slonimski (l), and by Ephrussi c’t al. in 19% (2), and has bwn documented in thcl intcwwGng 1.5 years b\- numerous inwstigators (F-14). It manifrsts itschlf as a block 011thr dcw~lopmc~nt of thcl ~11’s resI)irat ory capacity by glucose in high CY~CPII1ration. ‘1s a corollary, its CoIiv(‘rs(‘~r(ll(‘asc from reprwsion, or dcreprcssioli-rc~sult,s t’ronl t lw abwnw or rcwoval of this block: it owurs during gro\vth on nwior slowly fcr~nclnt~nblc carbc ~11 sources &her diwctly by th4r oxidation to CO2 or by such rwpiration subwlucwt to wrobic glycolysis, which COINv&s Fcrmcwtnbl(~ carbon sources to clthanol

Ilsed a haploid strain lX31Wl) (CU:tdel: obt :tiw(! from Prof. C. Avers. C’ells were grown at 30”(’ 011 a semisynt,hetic medium (3) using 1’ ( ~~LICOS~:IS (aart)on source lmless ot,herwise stated. ( ;rowth or cldtures was iriitiatcd at :t cell f’onc’(‘IlI r:ttion ()I lO~~lO”/ml while analysis of physiologic~:ll (.lI:lr:t(.teristics was started at -3 X 10”. ml.

(2, 1.5).

Our o\vn intcwst in this phenomenon is of standing (3, 5, 6, 13, l&IS), and is diwctlv rel:ttc>d to our conwrn \\ith the mode 01’ b&wthcGs of mitochondria in this organism, sinw wpression appears to affect a \vidts varic+y of mitochondrial proteins and oththr componwts, localizcld in all four mitochondrial compartments. It thus provides the invcsbigator \vith a ready m(‘ans of &udying the regulation of mlt~ochondrial dcwlopmcwt, or “differentiation.” E’urthermore, becauw t’h(l yeast cell on exponential gro\\-th on high glucose, even aerobically, satisfies all it,s cw’rgy requireme& by gly(*olysis and I~OIW by rrspiration, mitochondrial fun&ion-at, least 80 far as respiration and clncrgy transduction are concernedbocomc~disprwsablc. In this manner not only mitochondrial but) cvm cellular growth and division may hc studied under conditions \vhcn mit,ochondrial function, or the system rwponsihlc for mitochondrial gcnc rcplication :md expression, has been completely blockc~d by appropriate> inhibitors. It is the purpose of this communication to prcwnt~ a detailed description of the phenom(wo11of dewprcwion and to probe some of the factors that control it. long

RIETHOIX Crcllure c~otitlilioirs. A prototrophic commercial strain of Succharo,/l?/ces cerevisiae (var. Fleischmann) and a cytoplasmic pet,ite mutant derived from it, by ethiditun bromide treatment were used extensively. In one experiment a haploid strain (IL8-SC, 01his- try- p+CYE7~) and its petite mutant (IL8-8C,/l<5/3 p-Cal<“) obtained from Prof. P. P. Slonimski, were llsrd; in another experiment we

Daltc p,reuc/,/cr(io/,. We present nlost tit’ 01lr data by plotting total activity, riorm:rlizrd IO t IIC’ init ial value, against time. Total activity is 1~:1l(~bd:llc~tl hy mliltiplying specific activity (Iltlils lng bob mogenate protein) by the dry aeigtrl, 1111(11 c111. ture; it is proportional to llnits of rnzyrnc~ or cyfochrome ahsorbancc per ml of c~llltllrc~. h t’tlrv,x with :I positive slope indicates net s.vnthcsis (of ior a(‘tivation of preexisting) enzyme; :L urg;ltivc, slope indicatrs destrtlction (or inac*tivat 1011); 31rti :t slope of zero indicates IIO net synthesis. l.:ach OI these c~ircumstanc~rs was rncollnt(srcd tIltring t hcsr studies. III each figtire legend the initial specific activity for each parameter is prcsc~litc*tl so th:it, actual v:thlrs may t)r obtained if tlWircV1. VWquently we also preselit plots of spel.ific act ivily 01 specific, c.ontent,, agairr normalizcti :b t)eforc,, against time. 61lctr plots are valual)lr for ~~,rnp~ing preferential rate of forrnatiorl 01’ :tc’t ivat iota. .Iln/critsls. Acriflavinc, CU.osogl~~t:iric~ :~c.lti, at11imycin A9 chlorarnphellicol, cy~lohexituitlt~. C’J.I(Ichrome (’ (type I II 1, rthidilnn hronlitlf,, ~~~iotiomit,rotet,razolillnl violet (grade I), isocit rica :lc,itl (trisodillm salt), reduced ni~otillarrlitie-:ttlrllirle nicotinamide dirrucalrotlclc ph(lsdinrlcleotide, phatc, cis-oxalacetic acid, and phetlnzirlt~ nlethosldfate were purchased from Sigma (‘h(‘nli(.:11 (‘I). (;lus~~l:tsc~ was ptirrh:csrd from L’:ndo 1.:111- I’( ‘-

250

PERLMAN

AND TABLE

ACTIVITIES

MAHLER I

UNDER VARIOUS STATES OF REPRESSION Specific activityas *

Specific absorbancec

____ Conditions

loge 1% Glut log

0.5

lyc Glut

3.5

5% Glut

stat

170 Gal log 2y0 EtOH

0.5

1.0 log

3y0 Lac loge

1.0 0.6

Cyt ox NADH ox --

IrTADH: c

24 28.8 f2 118 k12.5 103 &6 135 f7.3 190 &23

2.6 1.8 A.22 54.9 f5 48 f3 68 f1.5 92 f9.5

9 6.3 f.4 114 f17 143 f6 160 +10 145 f45

succ:c

SDH

MDH

-~ .5 1.1: h.2 46 f9 18.7 f1.4 54.6 f9.3 65 t18

1.2 1.32 f.62 46 f7.8 45 f5.7 67 f9.3 -

a nmoles X min-’ X mg-I. b f Signs indicate standard error-all such values represent periments with each determination in duplicate.

-

GDH

b

Cl

2.8 3.4 A.6 38.2 f1.8

14.1 18.6 h2.7 83 k3.4

7.1 8.2 *1 21.9 jZ1.8

38.8 f2.5

56 &3

*2

aa

-~ 172 226 zk16 2930 &128 1401 f24 4430 *200 3500 f280

12 5.35 f1.2 113 h14.6 77 *12 303 *30 265 %55

the results

of three

-

20.5

to five separate

-10 13.2 f2 77 f3.8

61.6 f4.6

ex-

c Absorbance X mg-1 X 104. d Turbidity e Separate

to which ccl s were grown experiment.

prior

to analysis.

and 3H-labeled phenylalanine were purchased from New England Nuclear. All other

chemicals

were of reagent

grade.

RESULTS Standard

States

In order to obtain a clearer picture concerning the phenomenon of derepression we considered it essential, first of all, to measure the characteristic properties of cells of our strain of S. cerevisiue in a number of carefully defined standard states. We have restricted this quantitative analysis of enzyme and cytochrome levels to the whole-cell homogenate because we have been unable to devise a method for the routine isolation of mitochondrial fractions essentially free of other membranous contaminants in satisfactory yield from repressed or derepressing cultures. One cannot draw strong conclusions from comparisons of specific activities in fractions of undefined and variable purity. Therefore, we point out that these studies assume a mitochondrial localization of respiratory enzymes and cytochromes; our own and other studies of enzyme location in these repressed, dere-

pressed and petite yeast cells support the validity of this assumption (13, 18). In Table I we compare enzyme and cytochrome levels of cells in these steady states. It is seen, with the strain used in most of these studies, that levels of respiratory enzymes in aerobically grown cultures in exponential phase never fall below the limit of detection even in the presence of 5 % glucose. Such cells are reproducibly only slightly more repressed than are cells grown on 1% glucose; the former do have a considerably lower level of oxygen uptake (OU)4 than do clAbbreviations: Acr, Acriflavine, euflavine (2,8diamino-lo-methylacridinium chloride) ; anti a, Antimycin A; CAP, Chloramphenicol; cyt, Cytochrome (e.g., cyt c = cytochrome c, etc.); cyt ox, Cytochrome c oxidase (cytochrome c: 02 oxidoreductase, (EC 1.9.3.1); DW, Dry Weight; Etd Br, Ethidium bromide (3,s.diamino-5-ethyl-6phenylphenanthridinium bromide); F,, Mitochondrial ATPase (EC 3.6.1.4) ; GDH, L-glutamate dehydrogenase (EC 1.4.1.2); IDH, L,isocitrate dehydrogenase (EC 1.1.1.42) ; MDH, L-malate dehydrogenase (EC 1.1.1.37) ; NADH ox, NADH oxidase; NADH:c, NADH:cytochrome c reductase (antimycin-sensitive component) (EC 1.6.2.1); OU, Oxygen uptake (defined in text);

RECXJLATION

OF RESPIRATORY

the latter. Exponential growth on galactose yic4ds partially derepressed cultures, even aerobically when aerobic glycolysis is the predominant mode of its utilization. Cells allowed to derepress after growth on glucose are not physiologically or enzymatically identical with cells growing exponentially on c4hano1, even though ethanol is the major product of aerobic glycolysis and, therefore, is the predominant carbon and energy source to which t,hrl cells become adapted. Cells growing exponentially on ethanol or lactate (or glycclrol! not shown) are nearly identical in enzyme and cytochrome levels. It is clear that the extent of repression or thcl nt>t rate of enzyme synthesis may be set at a number of levels by external conditions; only anaerobic cult,ures grown on high glucose appear to exhibit a complete shutdown of respiratory cnzymc synthesis (1, 20). Presumably experiment’s using a chemostat (21-23) could be used to maintain the level of repression at any intermediate level desired unless t’he nature of the regulatory events is such as to permit only a limited number of discrete st’ates of gene expression. Properties of Repressed Mitochondria :l~itochondrial heterogeneity. It has been reported that “repressed mitochondria” are heterogeneous in their content of respiratory enzymes and, thus, represent a mixture of two main populations: one relatively poor in certain, but not all, respiratory enzymes, the other a great deal less so. The two populations were distinguished by their different buoyant densities; the light organelles, with relatively low enzymatic activities, were suggested to be precursors of the more dense, functional ones. The level of repression of the cell would then be reflected in the relative ratio of the two kinds of mitochondria (24-26). Wo have att’empted to extend these observations but have found that repressed cells yield a single homogeneous population of isolated mitochondria, which may be disOSCP, Oligomycin Sensitivity-Conferring Protein; Phe, Phenylalanine; SDH, Sllccinate dehydrogenase (EC 1.3.99.1); succ:c, Succinate:cytochrome c oxidoreductjase (EC 1.3.99.1); WCP, Whole cell protein.

L)EREPRESSION

2.51

tinguished from derepressed ones (which also consist of a single equally homogeneous population) by several of their physical properties. On elect#ron micrographs, espcGlly after serial sectioning, mitochondria in r+ pressed cells appear considerably larger, nlor’c irregular and highly articulated in shape than the rtrlativeli symmetrical CIIW’S swn in cells released from glucose repressiori or growing exponent ially 011 glycerol or lnctat e. Mitochondria isolatc>d from reprtssed cells were found to bc much larger thaII :irc their dcrc~prc~ssedctountclrparts (3). A\~ (‘spetted from this observation, t#hc rate -cdimentation profile of a mitochondrial frucrion isolated from a mixture of derepress;cstl1.~41s labeled with [%]Phe and repressed ~41s labeled w&h [3H]l’he (Fig. 1A and R) shows a marked difference in sedimr~ntation ratr> for t,he two particlr types. Thtb distribution ol’ the two isotopes reveals that each compon~~nt, consists of a single major species plus sinnll~ contaminating (or damaged) mat,c~rial; th(* region of overlap between the t,\vo nlnin peaks is quite small. In another expcrimcsnt, a mixed mitochondriwl fract,ion \vas a&~-zetl by isopycnic banding (Fig. I C:j; it (XII 1)~ seen that rcpresscd m&ochondria arc slightl> more dense than are derepressed OMY. -1 similar result has been obtained in anothf~r these t,wcj particle laboratory (27). Wherl types are isolated separatc>ly, :tn:tl.vz~~ hi* rate or isopycnic sediment)ation, and :\ssayed for the distribution of rcspirat orv 1’r1zymes, each exhibits a single peak of &ivit,y for cyt ox, SDH, succ:~’ and MDH (Fig. 2i. It is apparent from the experiment s describedsofar that, operationally, derepreAon of mitochondria leads to (1) an increw specific content of certain, but not. ne(0sariIy all, proteins of their inner membmne~;. (2) a diminution in mitochondrial size, anti (:j) an alteration in their buoyant dcn>itics, probably related to an altered phc~spholi~~iclto-prot#ein r&i0 of t’heir membranc>s. A number of difftlrcnt experimental attempts to demonstrate a precursor-product rclat iorlship between repressed and dereprc>s;sedn~it ochondria has yielded ambiguous results : our belief that such a relat,ionship exists iz IX& stered by the recent dcmonst~rntion of the retention of protein caomponents of an:~(‘r()bic

252

PERLMAN

promitochondria in the mitochondria produced after exposure of anaerobic cells to oxygen (28). Assessment of the relative contribution of mitochondrial differentiation (qualitative

AND

MAHLER

change in mitochondrial membrane composition) and proliferation (increase in mitochondrial mass per cell) to derepression requires a comparison of purified mitochondrial fractions isolated from fully repressed and

60

o--2

’ 9

1

’

17

Fraction

’ no.

Fraction

no,

FIG. 1. Sedimentation properties of isolated mitochondrial fractions. A, B. Rate sedimentation: A culture of repressed cells (A 600= 0.6) was placed in fresh medium containing 2Cc glucose and labeled with 1.5 mCi of [3H]phenylalanine for 2 hr. A derepressed (A~,00 = 3.4) culture was resuspended in fresh 2% ethanol medium and labeled with 50 PCi of [‘“Clphenylalanine for 2 hr. Both cell samples were then washed with distilled water and then mixed. The cells were converted to spheroplasts and a mitochondrial fraction was isolated by differential centrifugation as described in Methods. The fluffy layer was discarded and the tight pellet used for analysis by rate sedimentation on linear (15-55yc) sucrose gradients. Gradients were centrifuged (4°C) at 12,000 rpm to w2t = 96 X lo7 (A) and 212 X 10’ (B) using an SW-27 rotor in a Beckman L2 65B ultracentrifuge. Gradients were fractionated using an Isco fractionator and samples assayed for both isotopes on Whatman No. 3 filter discs; samples were corrected for spillover, quench, and counting efficiency as described previously (13). Dpm recovered were (A) 8.01 X 106 and (B) 10.7 X lo6 for [3H]phenylalanine and (A) 9.25 X lo4 and (B) 9.1 X lo4 for [l%]phenylalanine. C. Isopycnic banding. In an analogous experiment (using haploid strain D310-4D) the gradient was centrifuged at 63,OOOg for 1 hr. The slowly sedimenting material is observed in panels A and B bands with the main peak; it probably consists of damaged mitochondria and contaminating membranes. As noted previously (13, 18) most cellular membranes in aerobically grown yeast band at about 38-40y0 sucrose. The positions of peaks observed under these various conditions agrees with expectations based on the distributions of mitochondrial marker enzymes determined in separate experiments (cf. Fig. 2 and Ref. 13). 93,000 cpm 14C and 403,000 cpm 3H were recovered.

IbEGIJLATION

OF Itl~SPIlIATOl~Y

l~l51~ISPRl~:S~lOPi

DEREPRESSED

REPRESSED

15r %

SUCROSE

(w/w) \ ;....

. . . . ..*.**..‘.

IO-

/J&

5-I O=

I

1

1

I

I

L

I

20-

15-

,’ Y

0 ” 2

SDH

IO-

5-

8 o= 20-

15-

IO-

5I‘ OI

5

9

13

17

21

25

29

T

FRACTION

5

9

13

17

21

25

29

NO

Fro. 2. Sedimentation properties of enzyme activities in isolated mitochondrial fractions. Cultures of repressed or derepressed strain L)310-4D were grown under previous13 characterized conditions (13, 18). A mitochondrial fraction of each was prepared from a spheroplast lysate as described in Methods. A. A suspension (0.8 ml) of a derepressed mitochondrial fraction was sedimented into a 15-55yG sucrose gradient for 15 min at 33,OOOg. Gradient fractions were assayed for the enzymes indicated; each fraction is plotted and data points are connected by straight lines. B. A suspension (2.0 ml) of repressed mit,ochondrial fraction was sedimented into a E-557& sucrose gradient at 15,000g until ~21 = 284 X 10’. In both cases the marker enzymes coincided with each other and with the main protein peak (Am). Most of the A280units in fractions l-4 are due to bovine serum alhllmin which was added to stabilize t,he mitochondria.

dercpressed cells. While the latter may be readily obtained from spheroplast lysates using previously published procedures (13, 18), application of the same protocol to repressed cells J;ields a fraction of damaged mitochondria m low yield that is heavily contaminated by nonmitochondrial (unit) membrane fragments probably related to the plasma or cytoplasmic membrane sys-

tems. When repressed mit’ochondria arc isolated using dereprrssed ones as carrier (cf Fig. 1 A-C) much of the contaminating material is eliminated although w have no reason to believe that mitochondrial integrity has been enhanced. A possible source of tjhr difkulty in prcnparing fractions containing intact mitochondria from repressed cells is t,hc rnorphol-

254

PERLMAN

AND

ogy of the particle. Examination of serially sectioned repressed cells shows them to contain large, asymmetric mitochondrial structures that may well be more sensitive to damage during cell lysis and isolation. The same technique also provides an unambiguous measure of mitochondrial volume and hence of mitochondrial mass (58). Preliminary results indicate that for the strain used in this investigation the increase of total mitochondrial mass during derepression is 4-fold or less. Some mitochondrial components increase by a similar increment (Table I). It is, therefore, reasonable to conclude that these represent ‘Lconstitutive” components of the mitochondrial membranes of aerobic cells regardless of their state of repression, and, therefore, provide a measure of mitochondrial proliferation. However, since the content of many other mitochondrial components increases by a factor of lo-40 (Table I), we conclude that derepression also leads to extensive mitochondrial differentiation.

MAHLER

absorbance is decreased at 548, 553, and 603 nm corresponding to the oxidation of cytochromes c, cl, and au3 (Fig. 3C) ; shifts in the

Cytochromes in Repressed Cells and Mitochondria Because the cytochromes of cells growing aerobically under conditions of glucose repression have not been examined explicitly by other researchers, one must be cautious about the assignment of the absorbance maxima at 559 and 553 nm to cytochromes b and cl, respectively. Anaerobically grown wild-type and aerobically grown petite cells contain two other pigments (cyt bl and bz) that absorb in this region (29, 30). Both of them are characterized by a double absorption maximum at roughly 552 and 558 nm of about equal amplitude; the precise assignment of X,,, is complicated by the fact that not all spectra reported have been obtained at the temperature of liquid nitrogen. At this temperature a typical absorption spectrum of preparations of repressed mitochondria (obtained by spheroplast lysis followed by differential centrifugation) reveals peaks at 548, 553, 558, and 603 nm (Fig. 3). The same spectrum is obtained using chemical (dithionite) or enzymatic (succinate, NADH) reductants (Fig. 3A). When antimycin A is added to the mitochondrial suspension respiring on NADH or succinate,

1

500

!

I

550

600

Wavelength

“m

FIG. 3. Analysis of cytochrome spectra in repressed mitochondria and whole cells. Mitochondrial fraction. 2-mm path length, 4.6 mg protein per ml. A. Dithionite-reduced mitochondria: same as NADH or succinate reduced. B. Oxidized mitochondria, Hz02 added before freezing. C. Antimytin A-treated mitochondria: treated with anti-A plus NADH. D. Repressed whole-cell spectrum, reduced by dithionite plus glucose. Peaks indicated by the arrows (1) are not found in mitochondrial fractions or in derepressed cells. (2-mm path length, 29.6 mg protein per ml). Repressed cells were grown on 1% glucose to A600 = 0.6 (fully repressed; cf. Fig. 5). The mitochondrial fraction was isolated from spheroplast lysates by differential centrifugation and then washed twice before cytochrome analysis in 0.1 M phosphate, pH 7.4, made 30y0 in glycerol. The mitochondrial fraction was enriched 9- to 13-fold over the homogenate fraction in specific content of succ:c, SDH, IDH, cytochromes aaa, b, cl, and c.

RKGULATION

OF RESPIRATORY

Sorct region are in agreement with this COINelusion. Since electron transport betlveen cyt b and cl is blocked by antimycin A (31-33), cyt h should become fully reduced, and, in fact, the peak at 55~ nm behaves in this fashion. If the bulk of the absorbance at 553 and 55s nm were due to cyt bl and bz their dual peaks should bc unaffected by this treatment. The analysis just presented suggest,-: that cytochromes cl and b are components of mitochondrial preparations from repressed cells. Comparable results are also obtained with preparations of dcrepressed mitochondria and with repressed or dereprcwrd cells (using glucose or dithionite as th(> wductant). Thc~ absorption spectrum of repressed cells faxhibit s tn-o additional peaks not found in mitocl~ondrial fractions (Fig. 3D). One is found at 5i:j nm and remains unaltered when the cells are fully oxidized (after starvation in phosphate buffer for 1 hr at 30°C) while anothw broad band is found at around 535 nm. Siuw the pigments responsible are not loculizrsd in the mit’ochondria, no attempt has lw(~n made to characterize them. During derrprcwion both peaks disappear, apparcnt Iv due to diMon; their continued synthe& appears to s:top early during derepress1011,a,t the start, of the forment’ative phase (seesbr*lcn\-) (DW = 1.0 mg/ml).

The respiratory capacity per mg of cellular matwial (OU) of S. cwevisiae growing aerobicall>- using glucose (1%) as the sole source of carbon and energy may be estimated by the ability of washed cells suspended in phosphate to respire in the presence of added glucose (Fig. 4). It is found that OU is low and constant throughout the exponential phase, and t’h:\t an increase of roughly 4-fold occurs during early stationary phase. After the completion of’ this phase several additional ccl1 divisions occur until growth ceases, presurnal~ly due to the depletion of some essent*ial nutrient. This is the pat#tcrn first establishcd hp Ephrussi et al. (a), and since confirnwtl in many investigations. Quite commonl~ ()I: or an equivalent parameter, the respirat(Jr)quotient, (RQ, Q,) has been used as a mcasure of the respiratory capa-

2!jij

I~EltlSPKESSIOi\j IO’

1

IL’ 0

i

1

4

I

”

8

” Time

12

11

16

”

20

11

24

J

(hrs)

FIG. 4. Growth of Saccharornyces cerezjisiae ou 1% glucose (aerobic). Growth may be measured by any of the parameters shown; dry weight (I)W) is the most easily reproducible one and was used routinely except as noted. Initial values were 0.212, 45, 0.086, and 0.22 for Asoo, O.U., whole cell protein (WCP) and DW, respectively. The culture was initiated with derepressed cells (OIT = 140) at 0.00028 mg protein/ml so that eight mass doublings had occurred by to on the plot. Levels of enzymes (c.f. Fig. 5A and 6A) and OU cannot be explained by dilution of preexisting enzyme during eight generations of growth. The bold arrow (>2 hr) denotes the beginning of the fermentative stage of derepression while t,he thin arrow (>4 hr) denotes the beginning of the oxidntive stage of derepression (see text’).

bility and hence the level of repression of yeast cells (e.g., 1, 2, 7-11, 27). An analysis of the levels of a number of enzymes (membrane-bound, matrix and cytosol; respiratory and nonrespiratory) and of respiratory pigments reveals that OU alone does not provide an adequate description of the derepression phenomenon (Fig. 5A, B; GA, B). While the level of cyt ox parallels that of OU it can be seen that all other dcrepressiblc enzymes are released from repression well before the cells reach stationary phase, t’hat is, before the exhaustion of glucose from the medium-and before OU exhibits its characterist(ic increase. These

succ:c

3 A

TIME

I

I 8

(HRSI

,

NADH:c

,

16

I

NADH ox

12

I

0

I

,

20

b QMI”

0

I

4

,

I

8

,

I

12

I

1

16

!

,

20

FIG.

5. Increases in specific activity of parameters in cells growing on 1% glucose. A. Enzyme levels. Cells for which growth data are presented in Fig. 4 were assayed for enzyme content in cell-free homogenates. The double line at 4:s hr indicates the beginning of stationary phase. The enzyme levels were relatively constant for the first three points; during the next 2 hr of exponential growth, all activities except cyt ox, increased several-fold: this increase is termed the fermentative phase of derepression. During this period the cyt ox level decreased somewhat. In stationary phase, cyt ox level increased and all other enzymes continued to increase in specific activity. Initial values were 3.78, 203, 0.57, 1.28, 27.1, 1.46, and 5.8 nmoles. min-‘.rng-1 for GDH, MDH, SDH, succ:c, cyt oxI NADH:c, and NADH ox, respectively. Initial DW was0.223 mg/ml. B. Cytochrome levels. In a separate experiment samples were harvested periodically and analyzed for cytochrome content at the temperature of liquid nitrogen. For cyt c and aa a small increase occurs prior to stationary phase; all four cytochromes increase during stationary phase. Changes in cyt ua3 and cyt ox levels do not correlate in time; the cells pass through a period in which the ratio of cyt aaa/cyt ox activity is high. Apparently the stoichiometry of respiratory enzymes is not fixed. Spectra were obtained with cells suspended in 30y0 glycerola. M phosphate buffer, pH 7.4, at 13.7-18.5 mg cell protein/ml using A typical a 2.mm path length. Initial values (absorbance/mg cell protein X 104) were 3.32, 20.6, 7.11, and 11.1 for cyt ~3, b, cl, and c, respectively. spectrum for to is presented in Fig. 3 (D). The first three values for cyt c and c1 are only approximate due to an anomalous peak at 535-545 nm (cf.

r

b

SDH

I:E(:ULATION

OF KESPII:ATOI:Y

I)I~I:I~:l’I~EXSIOK

FIG. 6. Increases in total activity (per ml of culture) during growth on I(2 glucose. I)ata from Fig. 5 were calculated as total activity (cf. Methods) and replotted. A. Enzymes. The change in biosynthetic rate during the fermentative phase of derepression is dificult to determine in this kind of plot. It is clear that the most active rate of synthesis (per hour) is att’ained just prior to stationary phase for all enzymes assayed except for cayt, ox. B. Cytochromes. The most active rate of synthesis (per hour) is attained prior to St:+-

t,ionary phase.

enzymw remain at their low, repressed levels only during the midexponential phase] of grow%h (DW I;. ‘l’h(> following observations on derepression dcwribt~ thr results of many cxpcrimwts, some of which are shown in Figs. 5 and ti : t hcbformw displays changes in specific ctnzymcbactivity or cytochromc content, the lattclr t’heir incrraws in the tot’al culture. (1) ltcprrsscd cells (below DW = 1.0 mg/ ml) arc’ capabl(l of synthesizing all enzymes mc~as~rc~d and of maintaining them at the constant and low (wpressed) lcvrl shown in Tabl(b I. While it, is possible that this (rrsidual) activity is localizc~d in a minority of the wlls ill thcl culture or in a minority of the mitochondria, the considerations discussed abow (Icig. 1, 2) make this supposition highly unlikely.

(2) Dewprcssion of each enzymatic activity begins at a defined time (corwlatcd with parameters that measure cellular growth, such as DW). It is manifested by an incrwsr in t)he rate of diff crcntial cnzymc synt,twsisor activat)ion--until it’ reaches a IICW (dcrcbpressed) lw~l, again characterist,ic for ctach activity in time and amounts (3, 10, 12, 13). (3) The incrcaws in amount pw wll appear t’o fall into two groups: those in the first small (inwhasw in group arc’ wlatively specific activity <&fold) [OU, cyt ox, cyt aa8, cyt 0, cyt cl, and ATPast: (34, 35)j and considerably below those of the swond group which includes all oth(lr nctivitic>s and PXhibits increases by > 12.fold; for instanw, while cyt, h incrcaws only 2- to ?-fold, q-t b-linked c>nzymw \K\‘ADH ox, suw : c md NADH: c (antiLL wns . componwt)] itw-cwc: by a great wction).

&al

mow

(cf Tahlv

I arid ncd

258

PERLMAN

AND MAHLER

ox synthesis is also observed at that time after which both cyt ox and OU become rapidly derepressed. This apparent anomaly is not reflected in the biosynthetic patterns of other activities or cytochromes (including cyt aas) and thus cannot be due to any generalized inhibitory phenomenon. (5) The rate of synthesis of all other enzymes and cytochromes begins to increase beyond the fully repressed rate while the glucose concentration is still measurable and in this particular strain is no less than 0.4 %. We may call this the “fermentative” phase of derepression. While cyt ox is synthesized during repressed exponential growth, its derepression suffers an apparent brief interruption precisely during the fermentative stage. (6) As shown, derepression may be formally divided into a fermentative and an oxidative phase. These phases can be distinguished by the energy source used for biosynthetic events. Operationally this is done by measuring their response to antimycin A (see below). Therefore, the repressed state does not persist throughout exponential growth on 1% glucose; a more explicit and precise definition of this term is required in each particular instance. This stricture applies particularly to studies which use OU as the sole criterion of the state of repression. Regulation of Derepression

In the preceding sections we have described the properties of S. cerevisiae in several physiological steady states, as well as the derepression paradigm used in this laboratory for a number of years. We now proceed to examine the regulation of the phenomenon in some detail in an effort to understand the time and nature of any regulatory events involved. We do this by examining the results produced by a variety of treatments of repressed cells, growing exponentially on glucose, on their ability to initiate and maintain the various phases of derepression. The Role of Mitochondrial Gene Expression Efects of CAP. Chloramphenicol (CAP) is an inhibitor of mitoahondrial protein syn-

thesis but it inhibits neither mtDNA nor nDNA synthesis, and it does not interfere with processesthat depend on expression of nuclear genes (5, 6, 10, 16, 36-39). We have, therefore, used it to probe for possible regulatory effects of the mitochondrial translational system on derepression either in a direct fashion or by virtue of the products synthesized by it. Its addition to repressed cells prior to the onset of the fermentative phase of derepression shows them to be capable (Figs. 7, 8) of continued synthesis of most of the enzymes assayed, including succ:c and SDH (not shown) at rates close to the normal, isometric values. Formation of NADH : c and NADH ox also takes place but at a reduced rate. These cells are also capable of initiating the fermentative phase of derepression at the same time as those of the control culture but the derepression of all activities ceasesabruptly as soon as the culture enters stationary phase. This is the point at which the control culture undergoes its oxidative and final phase of derepression which depends on mitochondrial energy generation. Cytochrome oxidase is unusual in several respects. In contrast to the other activities, it is not subject to derepression during the fermentative phase and, therefore, the effect of CAP on this particuiar responsecannot be determined. However, during this period there appears a reproducible increase in the activity of the enzyme in CAP-treated cells, perhaps due to the integration and resultant activation of subunits of the enzyme synthesized in the cytoplasm into sites composed of or determined by products of the intramitochondrial system present in excess initially. In accord with this hypothesis is the observation that the increase in cyt ox activity is inhibited by cycloheximide, an inhibitor of protein synthesis on cytoplasmic ribosomes. A second anomaly is that when exposure of cells to CAP is continued into the respiratory phase of derepression, where the greatest increase in cyt ox activity is observed in control cells, this interference with mitochondrial protein synthesis leads not just to an inhibition but to an actual inactivation or destruction of this activity. An insight into possible mitochondrial contributions to cytochrome oxidase synthe-

REGULATION

OF RESPIRATORY

TIME

DEREPREHSION

(HRS)

FIG. 7. Effects of CAP on the biosynthesis of “soluble” and respiratory enzymes when added to repressed cells. CAP dissolved in methanol was added to a final concentration of 4 mg/ml to cultures growing on 1% glucose when A600 = 0.65 (repressed). At 1-hr intervals for 6 hr aliquots of CAP-treated (dashed line) and control cells (solid line) were chilled and harvested. Enzyme content was analyzed and converted to total units using w-holecell protein/ml of culture as a measure of growth. Initial specific activities were 0.175 mg/ml, 10.3, 1.5, 1.27, 1.59, 294, and 6.15 nmoles min+.mg+ for WCP, cyt ox, Ku’.kDI-I:c, SDH, succ:c, MDH, and GDH, respectively. In this experiment the fermentative phase of derepression began at 1 = 1 hr, while the oxidative phase began at t = 3 hr.

sis during the period immediately preceding the fermentative phase of derepression is provided by studies in which CAP is added to cells during midexponential growth on 1% glucose, when they are ordinarily capable of continued isometric synthesis of cyt ox and cyt au3 as well as of all the other markers tested. Under these conditions CAP affects their ability to elaborate cyt ox and cyt aa3 throughout and much more profoundly than that of any other component. In this experiment the fermentative phase of derepression only begins during the interval between the last two sets of data points (Fig. 8). Therefore, the inference is justified that the inhibition of the elaboration of cyt ox by CAP is not restricted to the oxidative phase of dere-

pression. Instead the marked effects observed (cf also Table II) suggest that mit,ochondrial protein synthesis, by the usual CAP-sensitive route, is present ‘in repressed cells. This process is required for the maintenance of the level characteristic for such cells of respiratory enzymes, especially cytochrome oxidase and perhaps certain cytochrome b-linked activities as well (for review see 39,40). As a corollary, these observations confirm the presence of all the components of the intramitochondrial system for protein synt’hesis even when its presence i.+ I\-holly gratuitous (38). We have also in\-rst#igated the effect on the biosynthesis of cytochrome c by such cells in the presence of C’.Yl’ (Table II). In this case, addition of the inhibitor

260

PERLMAN

AND

4

TIME

IHRSI

1FIG. 8. Effects of CAP on enzyme synthesis in repressed cells (exponential phase). Cells were grown on lyO glucose to AGo0 = 0.1 and transferred to sterile

l-liter

flasks (500 ml each). When -4600 =

0.2 (DW = 0.2 me/ml), CAP was added to 4 mg/ml to half of the flasks; all flasks were then incubated at 30°C and samples

were harvested periodically sample was then analyzed for enzyme content. The fermentative phase of derepression began during the last time interval; no samples entered the oxidative phase of derepression. Initial values were 0.3, 45, 4.8, 3.6, and 204 nmoles.min-l.mg-l for succ:c, cyt ox NADH ox, GDH, and MDH, respectively. Control: solid line; CAP-treated: dashed line. The small increase in cyt ox activity (per ml of culture) may not be due to net synthesis; activation of precursors, or stoichiometric alteration of preexisting enzyme may account for this increase. This interpretation is less satisfactory for the other enzymes assayed since the increases were much larger (5-23.fold). In other studies it has been shown that small increases in cyt ox activity are not accompanied by any increase in cyt ua3 (19).

during

a 3$hr

period. Each

MAHLER

component synthesized inside the mitochondria participates in the regulation of the derepression of this particular protein. When CAP is added to cells that have already completed the fermentative phase of derepression (at about the time of onset of derepression of cyt ox in control cultures, DW = 1.2 mg/ml), one now finds that the oxidative phase of derepression proceeds at a nearly normal rate for all activities assayed again except for cyt ox which remains completely inhibited (Fig. 9). Therefore, the ability to continue their derepression is clearly a reflection of the respiratory capacity achieved by the cells at the time of drug addition. Since cells in late exponential phase have completed the fermentative phase of derepression they are already capable of utilizing ethanol for energy supply while midexponential phase cells are not. On the other hand, cells that have been allowed to reach late exponential phase in the presence of CAP suffer from the added handicap of continued reduction in their cytochrome oxidase and effective energy transduction (41-43) by dilution and (possibly) turnover of the relatively small amounts of these proteins which were present at the time of drug addition. This dilution effect also accounts for the rapid cessation of all enzyme synthesis (including those provided by the cytosol) once these cultures become depleted of a fermentable carbon source. E$ects of Etd Br and Acr MtDNA and RNA synthesis are inhibited by the drugs ethidium bromide (Etd Br) and acriflavin (Acr) (44-46). Both drugs are mutagenic, the former being more effective in inducing the p+ to p- mutation (47-50). When these agents are added to repressed cells and respiratory enzyme levels monitored (Fig. 10) the effects observed are qualitatively similar to those obtained with CAP (cf Fig. 7). Although there is now some indiwith

cell growth

and

the biogenesis of cytosol

cation

of interference

and matrix

en-

zymes, the initiation of derepression of all enzymes other than cytochrome oxidase and

results in an overproduction of this component to a level exceeding that of completely

their

derepressed control cells (i.e., plateau in Figs. 5, 6). These results suggest

peded. At

values that a

continued

mentative treated

phase

synthesis

during

appears

relatively

the ferunim-

the concentrations used, cells with Etd Br for as little as 1 hr

RRGULATTON

OF RESPIItATOl
2ri1

I~I~:f~I~:P1~I:SSION

II

110 PETITES EXHIBIT THE FERMENTATIVE PHASE OF I)I:RJ.;PHI:SSION? Cell type Condition of growth Activity

Diploid

(Fleischmann)

P+ Ml)H 5“, (:luc lyO Glut 1”;) Glut l’;;, (;lr~c (21 )I3 5”;. (:luc 1’;; Glue 1”; Glut 1“; Glue cyt c 5% Glut 1’~;. Glut 1”; Glut 1“;. (:hlc

log log stat stat log log stat stat log log stat stat

+ CAPa

+ CAPa

+ CAPY

247 291 3480 1465 11.9 8.6 161.5 67 5.85 8.7 27.6 48.1

P.

280 429 1050

Haploid

p-CRP

P+ 494 756 3870

(iL5 649 874

9.4 10.1 33.4 -

21.8 3’2,a 193

24.6 55.4 65

12.5 13 37

29.9 28.4 37.2 -

“7 42.5 40.6

chCAP (final concentration = 4 me/ml) added to cells in log phase vested at the turbidities indicated in Table I.

produce only mutant progeny (
(IL-S-SC)

415 52x 7X6

(Am0= 0.2). .411 cells were bar-

ever, interference with mitochondrial gwe expression does affect both t’he court of derepression and the final maximal activity levels attainable since it arrests the claboration of a functional respiratory chain and with it support of the elaboration c~f all prot’eins dependent on it (oxidatiw phnst>). Role of Mitochondrial

Genornc

Ne have examined the question \vht:thc~r a stable petite mutant obtained from our wmmercial strain retains the ability to perform the fermentative phase of dereprcssion as measured by increases in specific content of cyt c, MDH, and GDH (Table II)-- such mutants are, of course, devoid of respiratory enzymes proper. The levels of enzymes and cyt c obtainable after cultures of this strain have fermented all glucose and reached stationary phase are comparable to thaw attained by CAP-inhibited pf culturw. Similar studies were then performed with a haploid strain of S. cerevisiae and two sta,ble pcitittr strains derived from it, one retairting gScwc,tic: information in its mtDn’A and t8he !,tjhor devoid of mtDXA and hence of all infornlation. While under t’hc conditions t~~q~loyed the repression of cyt c could not be obst~rwtl

262

PERLMAN

NAD-GDH

AND MAHLER

_._

A! 0

2

4

6

time. (hrs)

9. Effects of CAP on “cytosol” and respiratory enzyme synthesis when added to derepressing cells (Ano0 = 1.84). See legend to Fig. 7 for details. Drug was added at the beginning of the oxidative phase of derepression. Controls: dashed line; CAP-treated: solid line. Initial specific activities were 0.469 mg/ml, 14.6, 12.2, 16.3, 11.2, 301, 30.2, and 80 nmoles.min-l.mg-l for WCP, cyt ox, NADH:c, SDH, Succ:c, MDH, GDH, and OU, respectively. FIG.

with this p+ haploid, the synthesis of other enzymes appears sensitive to this control. The two petite mutants derived from it are capable of modest, and approximately equal, derepression upon exhaustion of glucose. These results provide additional and definitive evidence ruling out all participation of the mitochondrial genome and its products in the qualitative control of the early stages of dereoression. Role of Mitochondrial Function (Efects of Antimycin A) Since repressed cells contain all respiratory enzymes, it mav be postulated that this ca-

pacity of mitochondria for productive respiration even in midexponential phase itself controls the ability of the cells to undergo derepression. Thus, mitochondrial function may be required for derepression not as an energy source but in a regulatory manner perhaps by some positive feedback device such as translational control (e.g., through heme synthesis), as a template (52), or by some effect on the state of NAD(P)H oxidation. A test of this hypothesis is provided by the use of antimycin A. This inhibitor of the respiratory chain, acting between cyt b and cl (31-33), at a concentration of 1 pg/ml instantly inhibits respiration and growth of

REGULATION

OF I’LESPIRATOI1Y

1~15I:IZPI~I~:SSIOS

‘68

FIG. 10. Effects of transcriptional inhibitors on enzyme synthesis when added to rcpressed cells. The experimental design was the same as in the previous figures (7-Y). Ethidium bromide was added to 50 pM and acriflavin was added to 5 FM. Initial DW was 0.25 mg/ml. Initial values were 352, 0.63, 8.7, 10, and 45 nmoles.min-‘.rng-’ for MDH, succ:c, GDH, NADH ox, and cyt ox. Growth and mutagenesis data (yc p+) are presented in the upper center and right portion of the figure, respectively. Control: .--a-. ; Etd Br: --a--, for mutagenesis (solid line) data points are not shown (5C; p+ in 30 min); Acr: .~-o--,

derepresscd yeast cells, while it has no effect on the rate of growth of glycolysing or fcrmenting cells (19). The inhibitor was added to a culture of repressed cells, prior to the onset of derepression, and its effect on enzyme and cytochrome synthesis determined (Fig. llA, B). It is evident that all activities not directly inhibit.ed by the drug, including cyt ox, as well as cyt a,u3,cl, and c (which become more oxidized), and cytochromc b (which is rcduced) continue to be made so long as grow01 (on glucose) continues. Initiation of derepression takes place normally, and the extent of derepression (increase in SA) of all these entities agrees well with that obtained when the second phase of dcreprcssion is inhibitcd by the addition of CAP, Etd Br, or

Acr prior to the start of the first phase (I’igs. 7-10). Enzymatic activities depending on t>he cyt b-cl segment, of the respiratory cahain (e.g., NADH: and succinatc: cytochromc c reductasc) could not be assayed because of residual inhibitor, but again it is evident that derepression of the two cytochromes themselves did take place. In addition, because of the continued synthesis of cyt ox and cyt aas, the inference appears warranted that, a powerful inhibit,or of respiration does not by itself block mitochondrial protein synthesis, and that mitochondrial respiration as such is not, required for this process. Thcsc? results extend our earlier findings (18) and are in agreement, with those of Kovacs (53). The absence of any direct effect of antimycin A on mitochondrial protein synthesis has also

264

PERLMAN

AND MAHLER

Time

(hrs)

.FIG. 11. Effects of antimycin A on derepression when added to repressed cells. Experimental design was as in Fig. 8. When DW = 0.235 mg/ml, anti A was added to 1 pg/ml to half of the flasks (dashed line). A. Enzyme levels. Initial values were 4.36,306,0.8, and 45 nmoles.min-l.rng-1 for GDH, MDH, SDH, and cyt ox, respectively. Substantial deviation from the control was found only during the last time interval (stationary phrase). B. Cytochrome levels. Initial values were 5.6, 39.3, 22.4, and 17.9 absorbance units/mg cell protein (X lo*) for cyt uu3, b, c, and cl, respectively. Cell samples were suspended to 22-24 mg protein/ml and a 2-mm path length was used.

been demonstrated directly isolated mitochondria (54). Timing of the Initiating

by studies on Event

In previous sections we have shown that mid (e.g., .A600 = 0.2) and late exponential (e.g., A,,, = 0.8) phase cells growing on glucose while exhibiting similar OUs, enzyme, and cytochrome activities, differ in other respects, most fundamentally in the length of time required before they become derepressed. It is reasonable to ask, therefore, whether such cells would still differ in their ability to undergo derepression if the intervening growth period is eliminated? In other words, can we identify events or regulatory signals during these t,wo genera.t,ions that

may be required to “precondition” a cell for derepression? To test this possibility cells were grown to As00 = 0.2, concentrated 4-fold to Asoo = 0.8 by resuspension in a medium in which a second batch of cells had been grown to A 600 = 0.8 (“0.8 medium”). Growth and enzyme synthesis were then measured and compared to the same parameters in cells that had been grown to AGo0 = 0.S without interference. To control for possible artifacts caused by the manipulation of the Asoo = 0.2 culture, a batch of cells at A600 = 0.S was harvested and resuspended in the same “0.8 medium.” If intracellular physiological signals are indeed required for eventual derepression they would be expected to be con-

I:I5(;UI,,~TIOK

OF 1~f~:SPII:ATol:Y

1~I,l:I’Pl~fis~IoN

tion of som(l wtabolitc~ during gro\vtli in t hcs st’cadg stat{,.

oral cbarlior studiw on wlls gro\\-n under COILditiorls of c~xtcnsiw cataholitcx wprcssion is that their mitochondrin appear diffcwbnt from those in dcrcqnwwd cc4s not only in numbor but also in morphology (3, S, X-- 57). WV haw confirnwd and cxtclnded thrw (A)wrvations by t’licb USC’of serial wct,ions ;I tkti lvill ,show that, in fact, such cells of th(x strain used in the>currcwt study contain only a fw (l-5) highl!. branchcvl and articulal cd writochondria ctxtcknding thrtqhor~t the) cantopl:lSm

Tome

lhrsl

FIG. 1’2. Test for accumulation of a “derepression factor” in the two generations preceding derepression. The experimental design is described in the text. Initial 1)W = 0.58 mg/ml. Initial specific. activities were 33, 0.93, and 255 nmoles. min-‘.mg-’ for cyt ox, suw:c, and MDH, respectively. Shaded ckles -solid lines, control sample; open circles-dashed line, 0.2 + 0.8 concentrated sample; open circales-dot-dashed line, 0.8 4 0.8 sample.

veyed mtl perhaps partially cxecutcd \vhilc cells undergo &cl critical two divisions just prior to the first, phase of derepression. 111 this c:w then th(l 4-fold concentrated wlls isolntc~tl in early exponential phase should experience a delay in dcrepression. It is sc(:11 from Fig. 1% that all three: culturrs bfw)mct dcrcprrsscd at th(h same timcb and to thrx same cst,cnt This cxpcrimwt also demonstrates that wll~ gro\\-n on glucose to midnxponcwtial phnw arc in a stclady state with wspcct t,o glw~w wprwsion and to the ability to undr~rgo dwzprcwion in rwponw to a lo\vc:ring (11’eArawllular glucose: conwntrat~ion. The signal for the init,iation of the first phase of dc~rcqwcssionis, thwcforc, cxwukd rapidly and tlf w:: not rquire the: gradual accumula-

(58).

7’hC)

aWJUUt

for

d)OUt,

OUP-

third th volu~~~~(and mass fraction) ot that found in dwtpwssc~d wlls (<3 instwd ot 10 “;‘) and thrGr awrago volumc~ (1 ~111:~)is at lwst 10 timw gwatcbr than that of &rc~prcswtl mito~hondria ill situ. The l:tt.tcr ;Ir(t highly symmckriwl in sh:q)(b, clew to sphcrical or prol:ttt~ ellipsoids of rrvol~~tioti (diumcter or 1lliijor axis = O..j ~111)\\-ith ail :ivcwg~~ VOhUnC~

Of’ 0.5

pm:‘.

rhSc’

~~OllS~d~~~~l~~O~l~

h~{d

to t\vo important cwiclusioirs. l’hc usu:~l mitochondri:~l prcqwation fronr tqwswtl cells, containing Iwticlcs \vith tlw ~~onmon, roughl!. sphoric:~l mit OchOlldrk~ nlorpholog> usualI>- aw&tctl \vitli Iilitoc‘lioldrirr~ must itwlf rq)rcwM an artifact (.5!)). :\I] propcqtiw, particularly h~tlrotl~ri:LnIi(, onw (,T(I Fig. l), usually :tscribwl to such partklw ;fw valid only. f'or thaw isolatchd cnt,it ios, fornlc~l by a11 altwation and Iwrhaps disrl~1)tiotl ot th(b t,rw irlt r:wllul:w I~trti~l~~.

266

PERLMAN

AND MAHLER

fact, be higher during repression than in its absence. As a corollary to the presence in repressed mitochondria of a variety of respiratory activities, some with a requirement for mitochondrial translation, are the results obtained with CAP, Etd Br, and Acr. The effects observed (Fig. 7, Table II) as well as those described by Tzagoloff and his collaborators (4&43) suggest that inhibition of mitochondrial translation and/or transcription produces a further alteration in mitochondria and their components, even under permissive conditions when mitochondrial energy supply is dispensable, and the interference with mitochondrial biosynthetic activities has no detectable effect on cellular growth. The Derepression Phenomenon Levels of various activities. The studies presented here (Table I), as well as others reported in the literature, leave little doubt that the level of catabolite repression of cell populations growing exponentially in batch culture, or continuously in a chemostat, is itself subject to regulation by genetic and environmental factors. Thus, for the strains used in this and our previous investigations, a glucose concentration of 1% is both necessary and sufficient to produce close to the maximal level of repression a,ttainable. The same considerations appear to hold for the strains of S. cerevisiae used by Beck and von Meyenburg in their experiments (22). In contrast the strains of S. cerevisiae studied by Linnane and his collaborators (12, SO), by Mian et al. (23), and especially the S. carlsbergensis strain described by Bleeg et al. (61) appear to require much higher glucose concentrations before their maximal level of repression is attained. We have indications of similar behavior with some other strains of S. cerevisiae currently under study. On the other hand, a different strain of S. carlsbergensis investigated by Cartledge and Lloyd (62, 63) already undergoes derepression at a glucose concentration considerably higher than the one reported hem. Although growth rate on a given glucose concentration may be a contributing factor, it would not appear to be a major one since doubling time appears to be of the order of 60 min for most of the strains reported. It is not only

the concentration of catabolite required for maximal repression, but also the level of repression actually attainable under these conditions that appears to be strongly strain dependent (see e.g., Table II). Although none of the studies we are aware of has measured as complete a battery of cellular activities and concentrations as is provided by Table I, there is enough overlap in the studies cited to indicate the validity of this inference. The cells described here simply do not appear to be subject to as intense a repression of cytochrome oxidase, for example, as are some others. Finally, the same appears to hold also for the levels characteristic of maximally derepressed cells as reported by a much larger number of investigators (4, 7, 8, 19-13, 34, 35, 62, 63). The net effect is that the incremental increase of e.g., cytochrome oxidase between the two steady states is itself subject to extreme variability. Although not a subject of these particular studies, another observation appears pertinent at this point: the change in number and morphology of mitochondria found in strongly repressed and derepressed cells also appears to be strain dependent. Of the various strains examined by us (58) the one described here exhibits the most extreme pattern of transition: from one to three highly branched and articulated mitochondria pervading virtually the whole cytoplasm (see also 64) to 100 or so roughly spherical particles with an average diameter of 0.5 pm localized mainly around the cell periphery (cf also 3, 51, 55, 56, 59). The level of repression can be controlled physiologically by varying either the nature of the fermentable carbon source (e.g., the substitution of maltose, mellobiose, or galactose for glucose (7, 8, 18, 65) or the glucose concentration in continuous culture (23). Both paradigms also result in a decrease in growth rate, but in all likelihood the probable determinant governing this parameter as well as the regulation of repression, is some function of the rate of flux of carbon through the sequence of glycolytic intermediates-and the level of other metabolites generated thereby. The question naturally arises whether at intermediate levels of repression the population of cells, or of mitochondria within any

oitc ~(‘11,it,self rcfiocts a series of states intermediatcb beti\-wrl the fully repressed and d(wprcwsed one or a mixture of these two state,:: in differ& proportion. Clearly this prohltxm deserves serious exploration at the morphometric lc!vcl, the only way in which a truly decisive answer can be obtained. We have csamincd :L limited number of cells growing exponentially on galactose (58). Their mitochondria appear to be intermcdia,te in type and \ve see no evidence for grcatw intru- or int’ercellular heterogeneity than is observod ml lactate. However, much mow w-ark with cells at] a varied and greater level of reprrssion I\-ill be needed before the prcublcm is solvod. Phases iii fmfinuous tlerepressim. Continuous dcwprwsion of cells growing batchwise on a limited concentration (usually -10 g pc’r liter of glucose has been a favorite paradigm for R study of the phenomenon (for recent rcviwvs see References 38, 39, 66) and has been so WXY~in the current investigation. Its result,+ have led to the conclusion that dcreprossion of a variety of intra- and estralilitochorldrinl activities is initiated well Iwfow ~~11shave ceased growing and duplicating at the rate characteristic of esponcntial gro\\-th. In fact, at a cell turbitlit?. corresponding to 0.8 (the point m:&~d by a bold arrow in Fig. 41, the stage when d(wpression cat1 first be dctccted in our cxperimcnt,s, the glucose concentration cqunls 0.3 g pc’r lit,c,r and drops well below 1 g pw liter only, > 120 min later (at the point marked by tlw thin arrow in Fig. 4 and the double lint in the subsequent figures). During this jvholo period the cells are growing (for > one doubling) and are synthesizing various actjivitics at elevated levels by acrobit glycolysis >.ct dcrivc the bulk of their cncrgy supply by purely fermentative means (Fig. 11). We, therefore, propose to call t’his the fcrmentative phase of dcrcprcssion. The acwnulat~od c+hanol is utilized as a carbon sourc(~, and rcspiraGon as the required mode of c3ncrg.v gcncration, only in the second p1la.w wincidcnt with the sudden and discontinuous drop in growth rate. This phase, which 011 our figures is separated from the earliar one by the double lint:, might bc c:tlletl the oxidativc or respiratory phase of wllul:v and mitochondrial dcpreprcssion. It,

unlike the i‘cwnc~iitat ivc: 011c, is :~tw,lul(~l~ rtwpirxtor3 dependent. 011 mitoc~hondrial function. Of all t tic: activities c~xaniincd 1.)~. us, only cyt ochro~11c I’ oxidant and oxygw uptake appcw t 0 undergo tl(wprcwsiort solely during this rwpiratory phnsc:. 1-10~ cvcr, closf~ w~iniiutt ion (cl.g., l;ig. 6.1) slic)\~~ that tlw pllc:nomcr~on rosponsihl(~ is (~aplcts. 111 fact, t)lirrc: is an intwval during th(, fermc~lit,:rtivc: pliasc, \\-hwi eI:Aor:Lt,iwi 01 functional cvtochromo ~Jxid:lw [)rol):it)l! the rat e-lin&ng step for oxygen uptak(b :~h well-bcconic~s (~oriipl~ttcly rcpr(w(bd, jrisl when other activitiw have: :ltiaiIlcd t Iw conditions f’or maximal dercprwsioil. I II gcncrat, tlwir rates of iwwasc iw ~~oinp:w:~ blfl to tlicw charnc,t,cristic of the lath (t\; porwntial phase but, t,lic~~;result iii sigilific:IiIl incrcasw in spc>ciiic actwit!- lw(~:~~iw of 1li(a virtual ccwation of cellular growl h :It. :~ntl beyond this point. Subsequrntly ~1101’t,hoin, except for one wgnicnt, of tlw iwpirat orj chain (KAI>H: cytochromcb ,: wductaw), and perhaps cytochrorw b, actually lmdcrgo a transition charactcrizc~d by :I I( P\v(‘L’rat (’ of incrwsc characteristic J’or the* dlir:11 ion (:!do

illin

Scst,

nr

SO)

of

th

I’f:SpiI?LtOr)~

}jll:lSfb.

do WllS and t ticair rliitc)cliont~ri:r. coorclinat (11~ cluriug tl(,rc~l)rossic-)ri? In t8hcbw11sc that, (41s :lnd Illitoctlolltlri:I, exhibit idmtic!al f’h:lllgC!S ill IYlt(’ Of i’OrTn;ttiOIl and amount of tticsir cmustituerlts durilig (1~1 rcprcssion, t,ticbil~ts\v(‘r is clearly IIO. 2ZS 31 ready dkmssctl ttlcby fall into t,hwc~ (~I:Iss~~s: (i) cytcwliromc~ oxidasc:, (ii j YT\;hl )I-1 : c 2nd cyt,ochronlc: h, aid (iii) all othcw l(Qc~d. But fvfw \vit,liin an?. 011c cl:rss tlIc% :i(~ircil Clla~Ilgc~

268

PERLMAN

AND MAHLER

rate of formation of its individual members differs. Therefore, as we have already pointed out in our first study on the subject (3) cells and their mitochondria vary in their composition at different times during derepression. On a formal level at least, the phenomenon can be described as the insertion into a preexisting framework of separate entities at different rates. The same type of model has also been adduced much more recently for mitochondrial changes during aerobic adaptation (62). Derepression as mitochondrial differentiation. The results and their discussion so far indicate that macroscopically derepression of a cell population results in both qualitative and quantitative changes in their mitochondria. Some parameters, most notably mitochondrial mass and some of its “constitutive” entities such as certain cytochromes and oligomycin-sensitive ATPase, increase to about the same extent. These increases can reasonably be ascribed to some form of mitochondrial proliferation at the expense of other cellular entities, the synthesis of which is either slowed or stopped entirely. However, mitochondrial morphology and number undergo profound alterations during derepression and many of their most characteristic constituents are characterized by much greater but variable increases. Thus, cells and mitochondria can be properly said also to undergo differentiation coincident with, or as a result of derepression. At the microscopic or mechanistic level the interpretation is much less clear. Studies by VOID Meyenburg and Beck indicate that just at the beginning of the fermentative phase derepressing cells have reached their maximal level of budding, while by the end of this phase virtually all cells are free of buds (22). The same observation has been made with the strains used here and with others. However, the fermentative phase of derepression begins before this increase in the proportion of cells without buds. In addition, the possibility, that initially at least, derepression is limited to newly formed and growing buds and certainly its more extreme variant, that only these structures are even capable of initiating and undergoing the fermentative phase, can be ruled out by our finding that late

exponential phase cells growing on gIycero1 (a nonfermentable carbon source) also accumulate as single, unbudded cells. Requirements for ancl Regulation of Derepression At one time or another a number of different entities and events have been suggested as being implicated in the initiation, maintenance or regulation of derepression and mitochondrial development. We shall examine several of them in turn. Respiratory competence. This type of hypothesis has taken two forms: a postulated requirement forpreexisting, competent (i.e., derepressed) mitochondrial structures or for their function connected with respiration and its attendant energy transduction. So. far as initiation is concerned, one form of this hypothesis can probably be ruled out on ultrastructural considerations alone. Mitochondria in repressed cells exhibit a morphology grossly different from that of the particles after derepression, and there is no evidence for the coexistence of both types at any stage (58). Even though there have been reports of the identification of more than one band bearing mitochondria1 markers on gradient or zonal sedimentation of disrupted yeast cells (24-26, 63) these may well represent different types of fragments or patches resulting from the disruption of the original intracellular st’ructures. In addition, the respiratory chain and the activities linked to it in fully repressed mitochondria probably is not identical in its quantitative details to that found in their fully derepressed counterparts. Certainly, whatever its precise composition, its function is not required for the initiation and maintenance of the fermentative phase of derepression (13, 53 and this paper). Thus, this hypothesis can be ruled out. Macromolecules synthesized by mitochonCuria. Little can be added other than a restatement of the experimental observations. The initiation and maintenance of the fermentative phase of derepression does not appear to depend on the supply of proteins and RNAs synthesized in and by the mitochondria. Similarly, once a sufficient number of mitochondria have been formed or convert’ed to provide an adequate energy supply

l:I3;U1,.2TIO~

OF I1I~:S1’II:ATOll\r’

for tlw rwpiratoq. phase, the latter can also 1~ ~nuintail~c~d~and eventually turned off -in t,lw abwnw of furt’her mitochondrial protcGn synthwis (Fig. 9). Barath and I is rcquircd, in addition to a rcyuircwc~nt8 for tlirh replication of nwlcar gc:ncs explicit in the original hypothesis, to awount for t)hc incrcrtsed levels of synthesis of sonw proteins during their derepression rc+ttiw to thc4r isometric rates during re~JlWSd, cxpowntial growth. Except for cytochronw c \I(: SW: no evidence for thr key pwdiction of thr: model: the owrproduct,ion in th(a prcwnce of blocks of mit,ochondrial transcription and translation of the C(qNJWntS so regulated. I\ m(Jrc critical twt’ of the 1nod~1 might be provided by a demowtration of dcwprcssion evc~n in the complctc~ abwnw of replication of nuclPar IDS,\ (cf 1~c.f.(is). Rwrnt, work using Yeumspm-a o’assa as the expwimcntal sgstcm has sholvn that,, in addition t(J thrw protein subunits of cytochrome (’ oxidasc, part or all of cytochrome 1Jis ulso a nlitochondrial gene product (7% 7.;). Data wrc prcscnt’ed which suggest t’hat t.he pool size of inactive mitochondrial product is small for the cytochromc oxidase polypeptide and large for the cytochrome b polypctptide. WP have rcport’ed previously (:U) using lacta~t! grown cells of S. cweuisiae, the rapid and complete i&ihiticJrl of cytoclwonw oa3 synthnsis by CAP and the simultaneous partial inhibit,ion of cytochromc b s~~nthwis. In addition our present findings of dc4~>-cLtl (partial) inhibition by CAP and Ettl 13r of thrb synthwis of cytochromc blil~kc~(l ;ictiviticbs and more wmpl&n inhihi-

I )Kl: I~:I’I:I~:SSION

“fi!,

Thr ,/aturc cd’ f//c acid si~q//als. 1.rlt’or.. tunatc~l~~, UT kno\v :l grcsat, dcal nlor(’ :11)u1 \vhat factors or ctntitics arc not rquirnd f01 dcwpwssirjrt than thch oriw t,hut arc. z\1though tliwca aw sonic’ suggestive bits ()i evidcnw t)hnt, implicatch 3 ,.S’-c~~lict .1.111’ (revicwd ill Ref. 6(i), they are far frcjnl ('OILvincing. I’or instanw, although t,hc c~qwrim&s of l“ang and Hutow (69) d(bmonstr:lt (~3 stimulation of dnrc~prcssic~n(coinc:idc:nt V,i t 11 aerobic :idayltation) by. this conlpound, its substitution by ot h(tr nwlcotidcs gave riw to similar c4fwts. In our 01vr1 clxpc>rimcnts d+ signed to tc,st for this typcl of stimulatiotl oi dereprcwion \\ith c~orwntratctl acw)bic~ (~~11s suspcbndctl irl high gluc:oa~, the rwults \v(w: at bwt allibiguous (70). WP and cjthcw (71 ,l arr cont>inuing this lirw of c5plor:ti ion.

1.

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