THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255. No. 6, Issue of March 25. pp. 2624-2627, 1980 Printed m U.S.A.

The Relation of Heme to Catalase Apoprotein Synthesis in Yeast*

(Received for publication, May 15, 1979)

Wolfgang Woloszczuk,$g David B. Sprinson,ST( and Helmut Ruis(( From the +Department of Biochemistry, Columbia University, College of Physicians and Surgeons, New York, New York 10032, and the lllnstitut fur Allgemeine Biochemie der Universitat Wien and Ludwig Boltzmann-Forschungsstelle fur Biochemie, Wien, Austria ~

The synthesis of two hemoproteins, catalase A and catalase T, was studied in mutants of Saccharomyces cerevisiae deficient in heme formation. These mutants can be grown on the end-product heme or on a heme precursor, or on ergosterol and Tween 80 (a source of oleic acid). It was found by immunoprecipitation that, in the presence of heme, catalases A and T were present in the mutants, but that in its absence (growth on ergosterol and Tween 80) the apoproteins of these en- zymes were not detectable. In contrast, cytochrome c peroxidase, and some of the subunits of cytochrome c oxidase are present in cells grown without heme (Saltz- gaber-Muller, J., and Schatz, G. (1978) J. Biol. Chem 253,305-310). Other evidence suggests that absence of catalase T apoprotein under heme-less conditions may be due to control by heme of apoprotein synthesis (G. Ammerer and H. Ruis, unpublished results), rather than increased proteolytic degradation.

Saccharomyces cerevisiae contains three proteins with cat- alase activity of which two are hemoproteins (1-3). Both heme-containing enzymes are absent when the organism is grown anaerobically on a fermentable carbon source, but are formed during oxygen adaptation (4, 5). Furthermore, assem- bly to the holoprotein but not biosynthesis of the apoprotein of catalase A is very sensitive to glucose repression (6). Little is known about the function of heme in regulating the synthe- sis of hemoproteins in yeast. Such a mechanism could play a role during oxygen adaptation or glucose repression. Hence, catalase T, a cytoplasmic protein which might also be present in the cell wall, and catalase A, a vacuolar enzyme, seem well suited for a study of heme function in the biosynthesis of extramitochondrial proteins.

The present study attempts to answer the question of whether the biosynthesis of catalases A and T is controlled by the prosthetic group heme. Several observations suggest that heme or one of its precursors might be involved in such a control mechanism: ( a ) heme is thought to be present in only limiting amounts or totally absent in anaerobically grown yeast since the enzyme protoporphyrinogen oxidase requires

* This work was supported by grants (to D. B. S.) from the American Cancer Society, the National Institutes of Health of the United States Public Health Service, and the National Science Foun- dation, and (to H. R.) from the Fonds zur Forderung der wissen- schaftlichen Forschung, Austria, and the Hochschuljubilaumsstiftung der Stadt Wien, Austria. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Present address, 11. Medizinische Universitatsklinik der Univer- sitat Wien, Wien, Austria. 1 Present address, the Roosevelt Hospital, 428 West 59th Street,

New York, N.Y., 10019.

oxygen for the conversion of protoporphyrinogen to protopor- phyrin (7-9); (b) heme controls the accumulation of apocyto- chrome c1 in yeast mitochondria (10); ( c ) heme is necessary for the accumulation and assembly of cytochrome c oxidase subunits (11); (d) it has been suggested that coproporphy- rinogen, which is accumulated during anaerobic growth, reg- ulates the biosynthesis of cytochrome c oxidase subunits ( 12). A series of heme-deficient mutants, defective at different steps of heme biosynthesis, was described recently (13). These strains are respiration-deficient and auxotrophic for oleic acid and ergosterol. If they are unable to synthesize uroporphyrin 111, they also require methionine. When grown in the presence of heme or an appropriate precursor, they are phenotypically almost identical to wild type. In the present study, these mutants, especially those lacking 6-aminolevulinic acid syn- thase and ferrochelatase (strains GLl and GL10, respectively), were used to inquire whether heme or one of its precursors controls the formation of catalase A or T, or both. It was found that both proteins are absent in mutant cells grown without heme.

EXPERIMENTAL PROCEDURES

Yeast Strains Used and Culture Conditions-All heme-deficient mutants of S. cerevisiae were derived from the haploid strain X2180 (gal2) and were described previously (13). The strains used were: GL1, GL4, GM, GL7, GL11, and GL10. Strain ts136 (rnalgall) (14, 15) was obtained from Dr. L. Hartwell, University of Washington, Seattle. Double mutants and new recombinants (gal+) were con- structed by random sporulation (16) from strains X2180 and ts136. The protease-deficient strains (17) 16A (a, leul, heml, prtl-8) and 16C (a, leul, heml, pep7-1) were a gift of Dr. E. Jones, Carnegie- Mellon University. The strains were maintained on Medium A (1% yeast extract, 2% Bacto-peptone, 2% glucose) solidified with 2% agar. Heme-deficient mutants were maintained on Medium A supple- mented with 0.5% Tween 80 and 20 pg/ml of ergosterol. Stock cultures were grown to stationary phase on Medium A containing Tween 80 and ergosterol, and kept refrigerated for up to 3 days. Intermediate cultures were inoculated at 1% from stock cultures and grown on Medium B (0.2% yeast extract, 0.04% CaCL, 0.05% NaCl, 0.06% MgClz- 6H20, 0.1% KH2P04, 0.1% NH,Cl, 5 pg/ml of FeCL, 0.3% glucose, 0.25% Tween 80, and 10 pg/ml of ergosterol), or on Medium C (same as Medium B, but containing 1% glucose, 0.5% Tween 8 0 , and 20 pg/ ml of ergosterol). When 1% galactose was substituted for glucose (in the gal+ strains), the amount of yeast extract in Medium B was raised to 0.3%.

After 18- to 24-h growth, the cultures had reached stationary phase (absorbance at 660 nm for respiration-deficient cells was approxi- mately 2.1 wheh grown on 0.3% glucose and 3.5 on 1% galactose; absorbance was 8 to 9 for respiration competent cells on 0.3% glucose). This intermediate culture was used to inoculate at 1% the experimen- tal culture on the same medium containing 50 to 500 pCi of ~-[4,5- 3H]leucine. In some experiments, 6-aminolevulinic acid (20 pg/ml) or heme (13 pg/ml) were added to the medium with or without omission of Tween 80 and ergosterol. When Tween 80 and ergosterol were omitted from the experimental culture, the inoculum was washed twice with distilled water before use. Cells were grown in a volume of 30 to 60 ml in 300-ml Klett flasks on a rotatory shaker at 3OoC (22-

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Relation of Heme to Catalase Apoprotein Synthesis in Yeast 2625

23°C for the temperature-sensitive strains) and growth was monitored turbidimetrically (Klett-Summerson photometer, red filter, 660 nm). In certain experiments, a semisynthetic medium was used containing 0.33% yeast nitrogen base without amino acids, 0.1% yeast extract, 0.5% or 1% glucose, and varying amounts of Tween 8 0 , ergosterol, and heme (as described under “Results”).

In each experiment, the cultures were checked for revertants by plating aliquots on ( a ) Medium A, (b) Medium A supplemented with 0.5% Tween 80 and 20 pg/ml of ergosterol, (c) Medium A containing 20 pg/ml of 6-aminolevulinic acid, (d) Medium A containing 13 pg/ml of heme. Levels of p - mutants were determined by plating aliquots on agar containing 1% yeast extract, 2% Bacto-peptone, 0.1% glucose, 2% ethanol, and 13 pg/ml of heme. When strains containing the hem1 mutation were tested, heme was replaced by 20 pg/ml of d-aminole- vulinic acid. Cultures were harvested by centrifugation and washed three times with 0.05% L-leucine in the cold. Aliquots of 35 to 140 mg wet weight were transferred into polypropylene micro test tubes (Eppendorff, 1.5 ml volume) and stored at -15OC.

Preparation of Extracts-The cells were suspended in 1 ml of homogenization medium consisting of 25 mM sodium barbiturate-HC1 buffer (pH 8.2). 0.15 M NaCl, 1% Triton X-100, 0.05% L-leucine and broken by a 2.5-min treatment with 0.4 g of glass beads (0.45 to 0.50 mm in diameter) in a Braun homogenizer equipped with an adaptor for micro test tubes. After centrifugation (10 min, 5000 X g), the supernatant solution was removed and the homogenization was re- peated. The combined supernatant solutions were centrifuged for 30 min at 175,000 X g in a Beckman 50 Ti rotor and filtered through a 0.22-pm membrane filter. Label incorporated into proteins was deter- mined by treating aliquots of the extracts with an excess of hot 10% trichloroacetic acid, removing proteins by centrifugation, and meas- uring the amount of nonprecipitable radioactivity.

Immunoprecipitation-Extracts from yeast cells were made up to 0.1% SDS‘ by addition of 10% SDS, and immunoprecipitated with specific antibody and Staphylococcus aureus cells as described by Kessler (18) with some modifications. The S. aureus preparation (18) was stored as described, pelleted by centrifugation (5 min, 2300 X g), and incubated for 30 min at room temperature in homogenization buffer containing 0.1% SDS. The bacteria were washed once and resuspended in the same buffer to give a 10% suspension. The y- globulin fractions containing specific antibodies against catalases A and T, respectively, were prepared as described earlier (4, 5) and stored at -15°C in small aliquots which were thawed and centrifuged for 30 min at 9OOO X g shortly before use. An appropriate amount of antibody was added (at least enough to precipitate 1 unit of enzyme as determined by titration with active catalase). After 15 min in an ice bath, the bacterial suspension was added (twice the volume of y- globulin solution), and 10 min later the mixture was centrifuged.

The pellets were washed three times with the same buffer and suspended in a dissociating buffer ( 5 0 mM Tris-HCI, pH 6.8, 2.5% SDS, 1% P-mercaptoethanol, 6 M urea; volume equal to the volume of y-globulin solution used for immunoprecipitation). After 5 min at room temperature, the samples were heated in a boiling water bath for 2 min, S. aureus cells were pelleted by centrifugation, and the supernatant solution was analyzed on 7.5% SDS-polyacrylamide slab gels (1.5 mm thick) using the discontinuous buffer system described by Laemmli (19). In experiments in which protease inhibitors were tested, they were added to all buffers immediately before homogeni- zation, and before the immunoprecipitate was washed and dissociated. The final concentrations were 1 mM diisopropyl fluorophosphate, 0.1 mM N-a-p-tosyl-L-lysine chloromethyl ketone. HCl, 1 mM L-1-tosy- lamine-2-phenylethyl chloromethyl ketone, and 0.1% aprotinin. Gels were impregnated with 2,5-diphenyloxazole (sometimes after staining with Coomassie blue), dried, and fluorographed at -90°C with sensi- tized x-ray fdm for 1 to 30 days (20).

Fingerprinting-After fluorography, the dried gels were moistened slightly with HzO. Areas containing radioactive proteins of interest were cut out, washed four times with dimethyl sulfoxide (30 min each time), once with water for 20 min, and then for 20 min with buffer (0.125 M Tris-HC1, pH 6.8, 1% SDS, 1 mM EDTA, 1% P-mercaptoeth- anol) at room temperature followed by 2 min in a boiling water bath. The gel slices were transferred into wells of a stacking gel and subjected to proteolysis by S. aureus V8 protease as described by Cleveland et al. (21).

Other Methods-Catalase activity was determined spectrophoto- metrically a t 240 nm (22). A unit is the amount of enzyme which decomposes 1 pmol of substrate per min. Catalases A and T were each

. _ _ _ ~ -

I The abbreviation used is: SDS, sodium dodecyl sulfate.

measured in supernatant solutions obtained from cell extracts by quantitative immunoprecipitation of the other protein (23). Control experiments with a mixture of catalase A and T antisera showed that at least 99% of total catalase activity was precipitated. Hence, im- munoprecipitations with single antisera were quantitative. Protein concentrations were estimated with Coomassie blue (3-250 as de- scribed by Bradford (24) with serum albumin as standard. Radioac- tivity was counted in a SL-30 scintillation counter (Intertechnique) using a toluene/Triton X-100 scintillator. Materials-~-[4,5-~H]Leucine ( 5 0 Ci/mmol) was purchased from

ICN Life Sciences Group, Irvine, calif., 5’. aureus V8 protease from Miles Laboratories (U.K.), and heme 3 X crystallized from Nutritional Biochemicals Corp., Cleveland, Ohio. 8-Aminolevulinic acid, ergos- terol, Tween 80, diisopropyl fluorophosphate, N-a-p-tosyl-L-lysine chloromethyl ketone. HCI, L-1-tosylamine-2-phenylethyl chloro- methyl ketone, and aprotinin were from Sigma Chemical Co., St. Louis, Mo. Coomassie blue G-250 was from Eastman Kodak, Roch- ester, N. Y., 2,5-diphenyloxazole (scintillation grade) from New Eng- land Nuclear, Boston, Mass., x-ray Nm (Kodak X-Omat R) from Litton Medical Systems, N. Y., and membrane filters (GSWP) were from Millipore Filter Corp., Bedford, Mass. Other materials were reagent grade obtained from commercial sources and used without purification.

RESULTS

Heme-deficient Cells Lack Detectable Amounts of Apo- catalases A and T-In wild type cells, the two catalases were determined by measuring their enzymic activity, and by la- beling yeast cells with tritiated L-leucine followed by immu- noprecipitation with specific antibodies. Active forms of the enzymes, as well as precursors and enzymically inactive forms, can be detected by these methods (4, 5). However, owing to nonspecific adsorption during immunoprecipitation (Table I, catalase A), the immunoprecipitates were further character- ized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In extracts of wild type cells, fluorog- raphy of the dried gels revealed the presence of catalases A and T together with some minor bands. Extracts of mutant cells were devoid of both catalase apoproteins (Fig. 1). Other bands which were present on fluorograms from mutant cells account for all of the immunoprecipitable radioactivity from such extracts. Especially in immunoprecipitates of anticata- lase A, one dominant band (“X”) was found consistently. It was attempted to clarify by fingerprint analysis whether this peptide was a breakdown product of catalase A or whether it was present in the immunoprecipitate as a contaminant. Con- clusive results could not be obtained owing to the small amounts of the polypeptide detected in the mutants. Attempts to increase sensitivity in our search for catalase apoproteins by increasing the specific radioactivity of labeled cells in- creased the activity of the immunoprecipitate. Upon electro- phoresis, the small amount of immunoprecipitable material proved to consist of many unidentifiable polypeptides, but apocatalases were not detected in any of the extracts from heme-deficient mutants (Fig. 1). Dilution tests with labeled catalases showed that 2 t o 3% of catalase T and 1 to 2% of catalase A protein present in wild type cells could have been detected by our method.

TABLE I Incorporation of radioactivity into proteins precipitable with

antibodies against catalases A and T

Yeast strain Immunoprecipitated with antibody against

Catalase A Catalase T %

X2180 0.069“ GLl(heml-3)b 0.014 0.0015 GLlO(hemS)* 0.016 0.0045

0.135

a Per cent of radioactivity incorporated into proteins from ~-[4,5- JH]leucine (see “Experimental Procedures”).

Grown on ergosterol and Tween 80.

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2626 Relation of Heme to Catalase Apoprotein Synthesis in Yeast

Our failure to detect catalase apoproteins could be due to their rapid degradation in the absence of heme. We therefore tested different protease inhibitors as well as double mutants carrying the hem1 mutation and lacking proteinase B, or proteinases B plus C (17). Differences between conditions of normal and restricted proteolysis in the pattern of immuno- precipitated proteins from labeled cell extracts could not be observed in gel electrophoresis.

To rule out that glucose repression in a particularly drastic form might be responsible for the absence of detectable cata- lase apoprotein, gal' strains were constructed from strains GLI and GL10, and strain ts136. The results obtained after immunoprecipitation of radioactively labeled extracts and electrophoretic characterization were the same as for cells grown on 0.3% glucose.

Heme-deficient Mutants Supplemented with Heme Contain Active Catalases A and T Regardless of Enzymic Block-To determine the ability of the different mutants to become phenotypically wild type, we tested conditions in which the first and last intermediates of the heme biosynthetic pathway, i.e., 8-aminolevulinic acid and heme, could be supplied in the medium. Strain GL1 (hem]), lacking 8-aminolevulinic acid synthase, can be supplied with 8-aminolevulinic acid or with heme from the medium. Other strains defective in heme biosynthesis form active cytochromes and catalases when supplemented with heme (13). In preliminary experiments, it was found that utilization of externally supplied heme by the mutants is possible only in the absence of Tween 80, which seems to interfere with uptake or utilization of heme. In experiments where strains were supplemented with heme, the

T-

A- X-

1 2 3 4 5 6 7 8 9 1 0 1

3

FIG. 1. SDS-polyacrylamide gel electrophoresis of immuno- precipitates from soluble extracts of yeast cells grown to stationary phase on medium B containing ~.-['H]leucine. After immunoprecipitation and electrophoresis, the slab gels were dried and fluorographed. Soluble extracts (1 ml. 2.05 mg of protein, 2.2 X 10" cpm/mg) from wild type cells (lanes I to 3) were immunoprecip- itated as described under "Experimental Procedures." Lane I , no antiserum was added prior to addition of the immunoadsorbent; Lane 2, 25 pl of immunoglobulin fraction containing anticatalase A was added; Lane 3, 25 pl of immunoglobulin fraction containing anticata- lase T was added. Extracts (1 ml each) from mutant cells (Lanes 4 to 1 1 ) were precipitated with 15 1.11 of immunoglobulin fraction containing anticatalase A ( 4 to 7) or anticatalase T (8 to 11). Lane 4, and Lane 8, extract from GLI (1.06 mg of protein, 5.2 X 10" cpm/mg); Lanes 5 and 9, extract from CL4 (1.14 mg of protein, 6.1 X 10" cpm/mg); Lanes 6 and IO, extract from GLll (1.16 mg of protein, 4.9 X 10" cpm/ mg); Lanes 7 and 11, extract from GLlO (1.05 mg of protein, 4.2 X IO" cpm/mg). Band X is discussed under "Results."

TABLE I1 Growth characteristics and catalase activities of hem- mutants Cells were inoculated at 1% from Medium A containing 0.58 Tween

80 and 20 pg/ml of ergosterol, and grown to stationary phase on Medium C or on the same medium which contained 13 pg/ml of heme instead of Tween 80 and ergosterol. The inoculum was washed twice with distilled water for growth in the presence of heme. All assays were performed on cells grown with heme. Mutant cells grown on ergosterol and Tween 80 had no catalase activity (not shown in table). A unit is the amount of enzyme which decomposes 1 pmol of substrate per min. An absorbance unit at 660 nm corresponds to 4.6 X 10' cells.

Absorbance at 660 nm Specific activity Yeast strain Tween 8o + Heme

ergosterol Catalase A Catalase T

units/mg X2180 25.1 23.0 0.27 0.62 GLl(hemZ-3) 6.0 9.3 0.031 0.010 GL7(hem3-6) 6.2 8.3 0.009 0.174 GLG(hem4- 1 ) 5.2 8.0 0.140 0.020 GL11 (hem6) 5.7 8.7 0.083 0.021

inoculum was therefore washed carefully to remove traces of the detergent. Active catalases A and T were found in all heme-supplemented strains (Table 11). It can be seen from the optical density reached by the cultures grown with heme that the mutants were able to utilize ethanol, Le. to synthesize hemoproteins. I t is unclear why amounts of catalases and ratios of catalase A to catalase T activity varied from mutant to mutant.

The electrophoretic properties of catalases A and T from supplemented mutant cells were compared with those from wild type cells. Strains GL1 and GLlO were grown on media containing tritiated L-leucine supplemented with S-aminole- vulinic acid (GL1) or heme (GL1 and GL10). Immunoprecip- itates were obtained from the soluble extracts with anticata- lases A and T. The electrophoretic mobility of the catalases from the different cells was the same as from an extract of wild type cells.

DISCUSSION

The results presented in this paper show that heme is necessary not only for the formation of active catalases but also for the synthesis or at least accumulation of the apopro- teins of catalases A and T. In one of the experiments described, it was found that heme-supplemented strain GLl which is defective in 6-aminolevulinic acid synthase contains both yeast catalases. This mutant does not accumulate any inter- mediates of heme biosynthesis. Hence, there appears to be no requirement for these intermediates in catalase synthesis.

Although our results have demonstrated with high sensitiv- ity that catalase apoproteins are absent from unsupplemented mutant cells, they do not allow any definite conclusion on the mechanism of the effect of heme on catalase levels. Heme could act either as a positive regulator of the synthesis of apocatalases or it could prevent rapid degradation of these enzyme precursors. Synthesis of the proteins could be con- trolled by their prosthetic group at the levels of translation or messenger RNA formation. I t is well known that heme regu- lates the biosynthesis of globin in reticulocytes by controlling the initiation of translation (25). Heme could also act in an as yet unknown manner on the formation of catalase messenger RNA, i.e. on transcription, messenger RNA maturation, or transport. Alternatively, heme could protect catalase proteins against attack by intracellular proteases. Mutants defective in heme biosynthesis may synthesize catalase apoproteins nor- mally, but these incomplete and enzymically inactive products may be removed quickly by the degradative system of the cells (26). Apoproteins of other hemoproteins such as cyto-

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Relation of Heme to Catalase Apoprotein Synthesis in Yeast 2627

chrome c peroxidase or cytochrome c oxidase are accumulated a t least to some extent in heme-less yeast mutants or in cells grown anaerobically, i.e. under conditions when heme is not available or present only in limiting amounts (11,27,28). The apparent difference between the latter results and those ob- tained in the present study could be explained most easily by assuming that apocatalases are more susceptible to proteolytic degradation than the other proteins.

However, several observations make such an interpretation somewhat questionable. ( a ) Our investigation has shown that mutants lacking some of the main proteases of S. cereuzsiae do not accumulate catalase apoproteins in the absence of heme. ( b ) Addition of a mixture of protease inhibitors during breakage of cells, immunoprecipitation, and dissociation of immunoprecipitates did not have any effect on our results. ( c ) Catalase apoproteins have been found to be fairly stable under other physiological conditions, i.e. during oxygen adaptation (4, 5) or glucose repression (6). It could be argued that, under such conditions these proteins may be stabilized by precursors of heme. However, these precursors are present in mutant GL10, which also does not accumulate catalase apoproteins.

A final solution to the problem of the mode of action of heme should come from studies dealing more directly with the question of whether catalase messenger RNAs are present and are translated in heme-deficient cells. A fist attempt in this direction has been made by constructing a strain carrying both hem1 and rnal mutations by crossing strain GLI with strain ts136 (results not shown). The mutation of the latter strain in the rnal locus is temperature-sensitive (15). At the restrictive temperature, the strain is presumed to be defective in the transport of messenger RNA from the nucleus to the cytoplasm. Supplementation of a hemlrnal double mutant with heme at the restrictive temperature after growth of the strain at permissive temperature should in principle furnish information on the presence of catalase messenger RNAs in cells grown in the absence of heme. However, insurmountable technical difficulties in these experiments have led to a failure of this approach. Further studies are currently being carried out on the in uitro translation of catalase messenger RNAs.' In the case of catalase T, it could be shown that its messenger RNA is absent from cells of strain GL1 grown in the absence of 8-aminolevulinic acid. Hence, heme appears to control catalase T at the level of messenger RNA synthesis or turn- over. The situation is less clear in the case of catalase A. The results presented in this paper could not eliminate the possi- bility that apocatalase A is synthesized but degraded rapidly in the absence of heme, and in uitro translation of messenger RNA has not yet yielded conclusive results on the presence of catalase A messenger RNA in heme-less mutants.

Although there are obviously other reasons for elucidating the mechanism of control of formation of a hemoprotein by its prosthetic group, the experiments described in the present paper are important in connection with studies carried out on the regulation of catalase formation by oxygen (4, 5) and ' G. Ammerer and H. Ruis, unpublished results.

glucose (6). Oxygen is the main and perhaps obligatory elec- tron acceptor during heme biosynthesis. This pathway is also controlled by glucose repression. Our study shows therefore that any mechanism of oxygen or glucose regulation of cata- lase biosynthesis will have to take into account the fact that heme may be necessary for synthesis or accumulation of catalase apoproteins.

Acknowledgments-We are grateful to Dr. Edith G. Gollub for helpful discussions, to Dr. Elizabeth Jones for constructing the heme- protease-deficient yeast strains, to Dr. E. Hartter for supplying the antisera used in this study, and to Dr. L. Hartwell for strain ts136.

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W Woloszczuk, D B Sprinson and H RuisThe relation of heme to catalase apoprotein synthesis in yeast.

1980, 255:2624-2627.J. Biol. Chem. 

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