JOURNAL OF BACTERIOLOGY, July 1978, p. 54-610021-9193/78/0135-0054$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 135, No. 1

Printed in U.S.A.

Molecular Events During the Release of 6-AminolevulinateDehydratase from Catabolite Repressiont

HENRY R. MAHLER* AND CHI-CHUNG LIN

Department of Chemistry and the Program in Molecular and Cellular Biology, Indiana University,Bloomington, Indiana 47401

Received for publication 6 February 1978

Transfer of exponential-phase cells of Saccharomyces cerevisiae, previouslygrown in 2% glucose, to a derepression medium resulted in a prompt increase inthe level of 8-aminolevulinate dehydratase, the rate-limiting enzyme of hemebiosynthesis under these conditions. This derepression exhibited a lag of 35 minat 230C and required the participation of both RNA and protein syntheses.Dissection of the molecular events during this lag period disclosed that RNAsynthesis, rnal gene function (messenger RNA transport from nucleus to cytosol),and initiation of protein synthesis were completed within less than 10, 18, and 24min, respectively. The potential regulation of derepression by mitochondrial gene

products and mitochondrial function was probed by means of a series of isogenic,respiration-deficient (rho-, pet-, and mit-) mutants; no such regulation was found.

Cells of Saccharomyces cerevisiae and otheryeasts are subject to catabolite repression byglucose and other sugars. This phenomenon andits converse, the release from such repression (orderepression), bring into play a comprehensiveregulatory program involving a large number ofactivities localized in various intracellular andintramitochondrial structures and compart-ments. For this reason derepression has receiveda great deal of attention (for reviews see 20, 22,and 37) since its first detailed characterizationover 20 years ago (39). However, in spite of alarge number of investigations of enzyme levelsunder various degrees of repression and rates oftransition between them (e.g., 7, 11, 13, 29, 34,37), to our knowledge there have been no de-tailed studies that deal either with the kineticsof the synthesis ofany single enzyme subsequentto release from repression or with those of thevarious synthetic events that must supervene inits production. This investigation attempts tocorrect this deficiency using as its object the 8-aminolevulinate (alv) dehydratase (porphobili-nogen synthetase, 5-aminolevulinate hydrolase,EC 4.2.1.24), the rate-limiting enzyme in thebiosynthesis of heme during the initial stages ofderepression (14, 18). To do so we have used aparadigm for derepression (23, 27) that involvesthe transfer of exponential-phase cells from astrongly repressing to a derepressing medium,which not only permits release from repressionto take place in the virtual absence of any cel-

t Publication no. 3133 from the Department of Chemistry,Indiana University, Bloomington, IN 47401.

lular proliferation, but also allows rather precisemeasurements of the time course of the former.We have also tried to determine to what ex-

tent, if any, an intact mitochondrial genome andthe functions specified by it participate in theelaboration of the enzyme and its regulation.

MATERIALS AND METHODSYeast strains and media. The following strains

were kindly provided by L. H. Hartwell: A364A (rho'a adel ade2 ural hisl lys2 gall), ts-136 (a mutant ofA364A, thermosensitive in the locus rnal), and ts-187(a similar mutant with a thermosensitive lesion in thelocus prtl). Strain ID41-6/161 (rho+ a adel lysl w-chlr ery' olil r oli2 parl r), from our collection, was theparent of a rho& derivative (obtained by ethidiumbromide mutagenesis) and of the five mit- mutants (4,41, 42) used in this investigation: M44 (an oxi3 mu-tant), M70 (another oxi3 mutant), EM17 and M113(two cob2 mutants), and E65 (a regulatory mutantmapping between oxil and chl). Descriptions of theisolation, characterization, and phenotypic propertiesof these mutants have been published elsewhere (24,26).The medium used for the growth and maintenance

of all these strains was YEPD, containing (grams perliter): yeast extract (Difco), 10; peptone (Difco), 20;and glucose, 20. Derepression medium was either YM-1 (8), supplemented with 2.5 g of glucose plus 30 g ofethanol per liter (YMDE), or yeast extract-peptonesupplemented with 2.5 g of glucose plus 10 g of galac-tose per liter (YPDG).Derepression and enzyme measurements. Cells

were grown in YEPD, usually at 30°C (except forexperiments with the temperature-sensitive mutants,when we used the permissive temperature of 23°C),from an inoculum of<10e celLs per ml to an absorbancy

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VOL. 135, 1978

at 600 nm of 0.40 (Zeiss PMQII), corresponding to 1.5x 107 cells per ml. They were then harvested, washed,concentrated, and suspended in a derepression me-dium as previously described (23, 27). At the varioustimes and temperatures indicated in the different ex-periments, cell samples were withdrawn, mixed withcrushed ice, and kept at 0°C until the end of theexperiment. They were then broken with glass beads(23, 27), the homogenate was centrifuged at 20,000 xg for 15 min, and the supernatant cytosol was used forthe determination of enzyme activity (32) as follows:porphobilinogen was formed at 370C in 2.0 ml ofreaction mixture containing 300 ,umol of tris-(hydroxymethyl)aminomethane buffer (pH 8.5), 150,umol of KCl, 10 pmol of 8-aminolevulinic acid, and 15pimol of fl-mercaptoethanol plus extract. The reaction(which in preliminary experiments had been shown tobe linear with time and amount of extract added) wasstopped after 60 min by the addition of 0.4 ml of 25%trichloroacetic acid; 1.0 ml of Ehrlich mercury reagent(30) was then added per ml of supernatant from thereaction mixture, and absorbancy at 555 nm was de-termined after 15 min. The amount of porphobilinogenformed was calibrated against authentic standards. Alldeterminations were performed in triplicate and cor-rected for zero time controls. One unit is defined as 1nmol of porphobilinogen produced in 60 min at 370C.Protein was determined by the method of Lowry et al.(21). Under the conditions described, the amount ofcytosol protein in the kinetic experiments equaled 2.0+ 0.1 mg/ml.

Materials. 8-Aminolevulinic acid and porphobili-nogen were obtained from Sigma Chemical Co.Lomofungin was kindly provided by G. B. Whitfield ofthe Upjohn Co., Kalamazoo, Mich.

RESULTSOverall characteristics of alv dehydra-

tase. Results of preliminary experiments indi-cated that unlike alv synthase, which was notreleased from repression for more than 2 h aftertransfer to the derepression medium (27), alvdehydratase reached maxmally derepressed lev-els within 60 min at 300C. With both strains,A364A and ID41-6/161, this amounted to 2300%of their fully repressed activities (1 unit x mg-'for both). This increase in activity coincidedwith the disappearance of alv accumulated inrepressed cells, which dropped from a total of490 (cytosolic) and 9.3 (mitochondrial) pmol per107 cells to undetectable levels within 60 minafter transfer. These results are consistent withthe supposition that alv dehydratase is indeedthe pace-maker enzyme in heme biosynthesis infungi (14, 18, 31).Derepression of alv dehydratase at var-

ious temperatures. Many of the experimentsto be reported were performed at 23 or 370C,which constitute the permissive and nonpermis-sive temperatures, respectively, for the two ther-mosensitive mutants used in some of our exper-iments, rather than at 300C. We therefore deter-

8-AMINOLEVULINATE DEHYDRATASE 55

mined the kinetics of the release of the enzymefrom catabolite repression at these three tem-peratures (Fig. 1). The data indicate that addi-tional active enzyme appeared after a lag of 15(370C), 20 (300C), and 35 (23°C) miin and in-creased linearly thereafter. This absence of apronounced temperature coefficient in theexpression of active enzyme is interesting andsuggests the participation of an unusual rate-limiting step in its elaboration.Participation of protein synthesis and

rnal and prtl gene products in alv dehy-dratase production. Addition of cyclohexi-mide (at 25 or 100 ,ug/ml) to the derepressionmedium completely blocked any subsequent in-crease in the activity of the enzyme. In twoexperiments at 300C it remained constant at therepressed level of 1.2 ± 0.08 units x mg-' for thefirst hour and then gradually declined over thenext 3 h to 0.85 + 0.12 (100 ,ug of inhibitor) or0.90 + 0.08 (25 ,ug) units x mg-'. These resultssuggest a half-life of -4 h for the base-line (re-pressed) enzyme. When chloramphenicol (4mg/ml) was used as an inhibitor of mitochon-drial protein synthesis, there was no effect on

0 10 20 30 40 50 60

TIME AFTER TRANSFER (min.)FIG. 1. Derepression of alv dehydratase upon

transfer ofrepressed cells to a nonrepressing medium.Cells of strain A364A were grown on YEPD at 30°Cto a density of 1.5 x 167 cells per ml, harvested bycentrifugation, washed, and suspended at five timesthat concentration in YMDE, prewarmed and main-tained at one of the temperatures indicated. Sampleswere withdrawn from the agitated cell suspension atthe times shown and kept at 0°C. Enzyme activitywas determined as described in the text.

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56 MAHLER AND LIN

either the rate or extent of derepression. Takenin conjunction, these findings can be interpretedto mean that enzyme production requires pro-tein synthesis on cytoplasmic but not mitochon-drial ribosomes.

Additionally, more conclusive evidence con-cerning this point has been obtained by the useof ts-136 and ts-187, two thermosensitive mu-tants of strain A364A. These strains were origi-nally isolated and characterized by Hartwell,McLaughlin, and their collaborators (8, 9) andcarry mutations in the rnal and prtl loci, re-spectively. Lesions in the former are presumed,but not definitely proven, to result in a defect inthe transfer ofRNA (including mRNA) from thenucleus to the cytosol (8), but do not interferewith either transcription or translation in mito-chondria (5, 25). Lesions in prtl lead to defectsin cytosolic (8, 9) but not mitochondrial (5)polypeptide chain initiation. When cells of thesetwo strains were grown and subjected to de-repression at 230C (Fig. 2), enzyme productionincreased linearly after an initial lag of 35 min,

2.0 _-

7 1.5

2x 1.0

2

>- 2.5>

2.0

LmJa- 1.5

0.5

0 10 20 30 40 50 60TIME AFTER TRANSFER (min)

FIG. 2. Derepression of alv dehydratase in ther-mosensitive mutants atpermissive and nonpermissivetemperatures. Experimental design was similar tothat described in the legend to Fig. 1 except for theuse of strains ts-136 and ts-187 and growth of cells inYEPD, which was at 23°C for both strains. Derepres-sion wasperformed in YMDE at the two temperaturesindicated.

and these two parameters were in each instancesimilar to those of wild-type cells under compa-rable conditions (Fig. 1). In contrast, instead ofan increase, we observed a small but significantdecline of base-line activity when cells, previ-ously grown at 230C, were placed in a derepres-sion medium kept at 370C. Thus the functionsblocked in ts-136 and ts-187 appear to be abso-lutely required for the formation of alv dehydra-tase.Timing of molecular events in release

from catabolite repression. The methodologyused is based on that of Kepes (15) and has beendescribed in detail by Lawther and Cooper (19)and Bossinger and Cooper (1). When applied tolomofungin, an effective inhibitor of nucleartranscription in yeast (17, 19), the protocol con-sisted of the addition of this inhibitor (1 ,ug/ml)to a number of flasks containing cells in de-repression medium (kept at either 30 or 2300)at different times after the addition of the con-centrated repressed cell suspension (taken as t= 0). The suspensions were then agitated for anadditional 45 min to permit the expression of thesynthetic capacity for alv dehydratase accumu-lated in the absence of inhibitor. Cells dere-pressed at the two temperatures in the absenceof inhibitor constituted the control.When applied to the thermosensitive mutants,

our protocol consisted of using derepressing cellsuspensions which were kept at 230C for varioustimes and then shifted up and maintained at370C for an additional 45 min. Control sampleswere maintained at 230C.

Results of such experiments are shown in Fig.3 to 5 and indicated that the production of activeenzyme required RNA transcription for 5 min at30°C (Fig. 3) and 10 min at 230C (Fig. 5), whilethe rnal gene product must function for 18 minat 230C (Fig. 4). Translation was initiated within24 min (Fig. 5), and, as already demonstrated,active enzyme appeared 11 min later.Participation of mitochondrial functions

and gene products in derepression. The bio-synthesis of a number of cytosolic enzymes andmetabolic sequences, subject to cataboliterepression, has been reported to be dependenton, or regulated by, functionally and/or geneti-cally competent mitochondria. Among them areenzymes for the catabolism of a number of car-bohydrates, the induction of which is either re-pressed (28, 35) or enhanced (10) in rho- ascompared to rho' cells, isocytochromes c (40),and catalase (H. Cross and H. Ruis, unpublisheddata). In some of these instances it has beenpossible to distinguish between mitochondrialfunctions specifically encoded in, or controlledby, the mitochondrial genome and general res-piratory competence (as distinct from ATP pro-

t. 6

ts 136 (rna 1)2.5-

ts 187 (prt 1)

1.0

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6-AMINOLEVULINATE DEHYDRATASE

0 4 8 12 16 20 30 35 40 45 50 S5 60 65MINUTES

FIG. 3. Determination of the execution time fortranscription at 30°C by means of lomofungin. Ex-perimental design was based on that described in (1).Cells of strain A364A were grown, harvested, andconcentrated as described in the legend to Fig. 1.They were then distributed into a number of flaskscontaining YMDE at 30°C, and lomofungin wasadded at the times indicated. Incubation was contin-ued for a further 45 min. Cells were then chilled andused for determination of enzymatic activity as de-scribed in the text (0). Samples without lomofunginconstituted the control (0). Control enzyme levels attimes corresponding to the experimental set are in-dicated by a heavy line; the raw data, showing theagreement between this experiment and that of Fig.1, are given by the dashed line.

duction) of the organelles (28).It therefore seemed of interest to compare the

rates and extent of derepression of a respiration-competent wild type with a series of isogenic(both isonuclear and isomitochondrial), respira-tion-deficient mutants derived from it. Theseconsisted of an rho- (which is devoid of func-tional mtDNA [16, 22]) and various miF mu-tants (harboring defined small lesions in differ-ept segments of mtDNA [4, 41, 42]). The miFmutants used were strain M44, an oxi3 mutantdficient in cytochrome oxidase (24, 26); M70,awiother oxi3 mutant with a different pattem ofnpitochondrially synthesized polypeptides (24,

EM17 and M113, both cob2 mutants defi-cient in cytochrome b and cytochrome oxidase(subunit I); and E65, a mutant in an apparentlynew locus mapping between oxil and cap (4)

and exhibiting a pleiotropic deficiency in a num-ber of polypeptides.The results of the first set of experiments are

summarized in Fig. 6, which shows the levels of

2.!

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C',

.5>-

..

( O.

s F/ ~~~~~~~rna5 '

0~~~~~~7

5//

.0

,5 111111111T,..5

- 0 4 8 12 16 20 30 35 40 45 50 SS 60 65

MINUTESFIG. 4. Determination ofthe execution time for the

function specified by the rnal gene (strain ts-136).Experimental design and representation was similarto that described in the legend to Fig. 3, except thatcells of strain ts-136 grown on YEPD at 23°C wereused. They were treated as before, transferred toYMDE, maintained at 23°C, shifted up to 37°C at thetimes shown, and permitted to express enzyme-form-ing capacity for an additional 45 min. Controls weremaintained at 23°C.

25 F

E

E

50F

wa-Cr

20 F

'5

1 0

05

0

0 4 8 12 16 20 24 28 35 40 45 50 55 60 65

MINUTES

FIG. 5. Determination of the execution time fortranscription and chain initiation at 23°C. Experi-mental design was as described in the legends to Fig.3 and 4 except that growth and exposure to lomofun-gin was at 23°C with strain A364A, and strain ts-187was used.

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58 MAHLER AND LIN

the enzyme in cells growing exponentially on anumber of carbon sources that differed in theextent of severity of catabolite repression theyproduced (2, 36). This paradigm, rather than themore usual comparison between glucose with anonfermentable carbon source, is made neces-sary by the inability of respiration-deficient cellsto utilize nonfernentable carbon sources. Fourdifferent conditions were used: growth to mid-exponential phase (4.5 x 107 cells per ml) on 5%glucose, on 1% raffinose, and on 1% galactose,plus cells grown on 1% galactose for an addi-tional doubling subsequent to that of the pre-vious sample. Two inferences can be drawn im-mediately: (i) respiratory incompetence is no barto either synthesis or derepression ofthe enzyme(compare glucose-grown cells of all strains, in-cluding the rho-, with those grown on all othercarbon sources); and (ii) as expected (2, 36), thegeneral order of repressivity at comparablestages of growth is glucose >> galactose > raf-finose. In all instances, enzyme levels in mit-mutants appeared lower than in the wild typeand somewhat higher than in rho-. A moredetailed analysis of the extent of derepression in1% galactose or raffinose relative to 5% glucosein wild-type, rho-, and mit- strains (Table 1)indicates that effective derepression is independ-ent of respiratory or genetic competence of themitochondria.

In the second set of experiments we comparedthe kinetics of derepression of the wild type withthat of the rho- and three of the mit- mutants.For this purpose we had to modify the nature ofthe derepression medium to the use of galactose(YPDG) rather than ethanol (YMDE) as a car-bon source. The results (Fig. 7) show that thelag period of rho- cells is longer, the kinetics are

J. BACTERIOL.

different, and the level of derepression is lowerthan that of wild type (100 versus 140%). Themit- mutants exhibited a lag similar to that ofwild-type cells, and the extent of their derepres-sion (133% for EM17, 150% for E70, and 125%for E65) also compares favorably with that ofwild type (140%).

DISCUSSIONThe evidence presented in this paper indicates

that when strongly catabolite-repressed cells ofS. cerevisiae are released from this constraint(i) alv dehydratase is the rate-limiting enzymein heme biosynthesis, and (ii) the kinetics of thisrelease can be followed readily; at 300C, produc-tion of new enzyme molecules began within 20min after transfer to a derepression medium.Using appropriate temperature-sensitive mu-

TABLE 1. Derepression coefficients for alvdehydratasea

S. cerevi- Mutation Affected Rb R"csiae strain function

ID41-6/161 Wild type None 0.50 1.6M113 mit7 aa3 0.69 1.6M44 mit- aa3 0.40 1.8M70 mitU aa3 0.45 1.2E65 mit- aa3 > b 0.88 1.8EM17 mit- b > aa3 1.0 1.0p rho- All 0.44 1.0

All strains were grown to a cell density of 4.5 x107 per ml. Affected function refers to mitochondrialcytochrome absent or reduced. Data are for enzymelevels determined as described in the legend to Fig. 6for cells grown on glucose (Glc), galactose (Gal), andraffinose (Raf), respectively.

b R = A(Gal-Glc)/Glc.R' = A(Raf-Glc)/Glc.

4.0

FIG. 6. Levels of alv dehydratase in wild-type and respiration-deficient mutant cells grown on differentcarbon sources. Cells of strain ID41-6/161 (wild type) and various isochromosomal mutant derivativesdescribed in the text were grown to a density of 4.5 x 167 cells per ml on 5% glucose (full bars), 1% galactose(empty bars), and 1% raffinose (cross-hatched bars); another sample was maintained on galactose to -9 x 167cellsper ml (stippled bars). Cells were harvested and washed, and enzyme levels were determined in triplicate;bars show standard deviations. Statistically significant variations (Student's t test) from wild type areindicated by one (P < 0.05) or two (P < 0.01) asterisks.

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V-AMINOLEVULINATE DEHYDRATASE 59

2 5

-cx

0'Ex'5Ec-

0

ii

wa-(I)

2 0

5

0

0 5

2 0

5

0

0 10 20 30 40 S0 60 70 80 90 ,00 ,0

TIME AFTER TRANSFER (MIN)FIG. 7. Kinetics of derepression of wild-type and respiration-deficient mutants. Experimental design was

similar to that described for Fig. 1, except for the strains, which were shown, and the use of YPDG asderepression medium. Results for wild type and rho& use the upper scale on the ordinate, and those for EMI 7,E70 (also called M70), and E65 use the lower.

tants we have shown that (iii) the rnal and prtlgene products must both participate in the pro-duction of new enzyme. Together with the re-sults of experiments with cycloheximide andlomofungin, these results suggest that enzymeformation in response to derepression requiresthe participation of both nuclear transcriptionand cytosolic translation.

One of our aims has been to establish thetimes subsequent to release from repression atwhich certain events involving macromolecularbiosynthesis must be completed. At 230C these"execution times" (1) are 10 min for nuclearRNA synthesis, 18 min for the function exercisedby the rnal gene product, 24 min for the initia-tion of protein synthesis, and 35 min for theappearance of active enzyme. To our knowledgethis represents the first dissection of the se-quence of macromolecular events occurring dur-ing the derepression of a catabolite-sensitive en-zyme in S. cerevisiae. They are to be comparedwith 1, 4, 9.5, and 13 min for the analogousevents during the induction at 220C of allopha-nate hydrolase upon the addition of urea (1).Among the more prominent entities subject to

regulation by catabolite repression are variousmitochondrial structures and functions, includ-ing their cytochromes (13, 33, 36, 40). Cyto-

chromes, of course, are hemoproteins, the syn-thesis of which may be regulated by alv dehy-dratase (6, 18, 38, 39). Thus a hypothetical reg-ulation of the derepression of the latter, in turn,by mitochondrial functions might provide anelegant regulatory feedback device.However, the interpretation of the experi-

ments comparing derepression in respiration-proficient and respiration-deficient strains is rel-atively straightforward in ruling out this hypoth-esis. They lead to the inference that mitochon-drial function, or an intact mitochondrial ge-nome and the products of its expression, are notrequired for effective derepression of the dehy-dratase.

ACKNOWLEDGMENTSWe thank L. H. Hartwell for providing the thermosensitive

mutant strains and G. B. Whitfield of Upjohn Co. for thesample of lomofungin used in these studies. We are alsoindebted to P. S. Perlman of Ohio State University andDonald Miller and Deborah Hanson of this research group forcollaboration and many helpful discussions concerning themit- mutants.

These investigations were supported by Public Health Ser-vice grant GM-12228 from the National Institute of GeneralMedical Sciences. H.R.M. is the recipient of Research CareerDevelopment Award K06-GM-5060 from the same Institute.

LITERATURE CITED1. Bossinger, J., and T. G. Cooper. 1976. Sequence of

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molecular events involved in induction of allophanatehydrolase. J. Bacteriol. 126:198-204.

2. De Deken, R. H. 1966. The Crabtree effect: a regulatorysystem in yeast. J. Gen. Microbiol. 44:149-156.

3. De Deken, R. H. 1966. The Crabtree effect and its relationto the petite mutation. J. Gen. Microbiol. 44:157-166.

4. Dujon, B., A. M. Colson, and P. P. Slonimlki. 1977.The mitochondrial genetic map of Saccharomyces cer-evisiae: compilation of mutations, genes, genetic andphysical maps, p. 579-669. In W. Bandlow, R. J.Schweyen, K. Wolf, and F. Kaudewitz (ed.), Mitochon-dria 1977. De Gruyter, Berlin.

5. Feldman, F., and H. R. Mahler. 1974. Mitochondrialbiogenesis: retention of terminal formyl methionine inmembrane proteins and regulation of their synthesis. J.Biol. Chem. 249:3702-3709.

6. Gollub, E. G., K. P. Liu, J. Dayam, M. Adlersberg,and D. B. Sprinson. 1971. Yeast mutants deficient inheme biosynthesis and a heme mutant additionallyblocked in cyclization of 2,3-oxidosqualene. J. Biol.Chem. 252:2846-2854.

7. Haarasilta, S., and E. Oura. 1975. On the activity andregulation of anaplerotic and gluconeogenetic enzymesduring the growth process of bakers' yeast. Eur. J.Biochem. 52:1-7.

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10. Haussmann, P., and F. K. Zimmermann. 1976. Therole of mitochondria in carbon catabolite repression inyeast. Mol. Gen. Genet. 148:205-211.

11. Holzer, H. 1976. Catabolite inactivation in yeast. TrendsBiochem. Sci. 1:178-181.

12. Hutchison, H. T., L. H. Hartwell, and C. S. Mc-Laughlin. 1969. Temperature-sensitive yeast mutantdefective in ribonucleic acid production. J. Bacteriol.99:807-814.

13. Jayaraman, J., C. Cotman, H. R. Mahler, and C. W.Sharp. 1966. Biochemical correlates of respiratory de-ficiency. VII. Glucose repression. Arch. Biochem. Bio-phys. 116:224-251.

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24. Mahler, H. R., T. Bilinski, D. Miller, D. Hanson, P.Perlin, and C. A. Demko. 1976. Respiration defi-ciernt muatants with intact mitochondrial genomes: cast-inga wWider net, p. 687-863. In T. Bucher, W. Neupert,W. Sebgld, And S. Werner (ed.), Genetics and biogenesisof chloroplasts and Initochondria. North-HollandBiomedical Prea, pAsterdam.

25. Mahler, H. R., and K,. Dawidowicz. 1973. Autonomy ofnitochondria ofSaccharomyces cerevisiae in their pro-duction of messenger RNA. Proc. Natl. Acad. Sci.U.S.A. 70:111-114.

26. Mahler, H. R., D. Hanson, D. Miller, T. Bilinski, D.M. Ellis, N. J. Mexander, and P. S. Perlman. 1977.Structural and regulatory mutations affecting mito-chondrl gone products, p. 345-370. In W. Bandlow, R.J. Schweyen, K. Wolf, and F. Kaudewitz (ed.), Mito-chondria 1977. De Gruyter, Berlin.

27. Mahler, H. R., and C. C. Lin. 1974. The derepression ofA-aminolevullnate synthetase in yeast. Biochem. Bio-phys. Res. Commun. 61:963-970.

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29. Matern, H., and H. Hlolzer. 1977. Catabolite inactivationof the galactose uptake system in yeast. J. Biol. Chem.252:6399-402.

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