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Proof of De Novo Synthesis of the Qa Enzymes of Neurospora Crassa During Induction

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Proof of De Novo Synthesis of the Qa Enzymes of Neurospora Crassa During Induction Proc. Nati. Acad. Sci. USA Vol. 74, No. 10, pp. 4256-4260, October 1977 Biochemistry Proof of de novo synthesis of the qa enzymes of Neurospora crassa during induction [catabolic dehydroquinase (5-dehydroquinate dehydratase)/quinate dehydrogenase/qa gene cluster/eukaryote gene regulation] WILLIAM R. REINERT AND NORMAN H. GILES Program in Genetics, Department of Zoology, University of Georgia, Athens, Georgia 30602 Contributed by Norman H. Giles, July 22, 1977 ABSTRACT In Neurospora crassa three inducible enzymes There is strong genetic evidence that the qa-1 gene encodes a are necessary to catabolize quinic acid to protocatechuic acid. regulatory protein which, in the presence of quinic acid, exerts The three genes encoding these enzymes are tightly linked on chromosome VII near methionine-7 (me-7). This qa cluster in- positive control over the synthesis of the enzymes encoded in cludes a fourth gene, qa-1, which encodes a regulatory protein the three adjacent structural genes (6, 10). The appearance of apparently exerting positive control over transcription of the enzyme activity is very specifically regulated. In the presence other three qa genes. However, an alternative hypothesis is that of a preferred carbon source, e.g., sucrose, the activities of all the qa-I protein simply activates preformed polypeptides de- three enzymes are quite low. Quinate as a sole carbon source rived from the three structural genes. The use of density labeling causes a coordinate induction of all three enzymes (9). This with D20 demonstrated conclusively that the qa enzymes are synthesized de novo only during induction on quinic acid. Na- increase in enzyme activity can be inhibited by the simulta- tive catabolic dehydroquinase (5-dehydroquinate dehydratase; neous addition of cycloheximide to the medium (ref. 5; J. A. 5-dehydroquinate hydro-lyase, EC 4.2.1.10)(a homopolymer of Hautala, unpublished data; this paper). This effect strongly ca 22 identical subunits) has a density of 1.2790 g/cm3 as de- suggests that all three enzymes are being synthesized de novo termined by centrifugation in a modified cesium chloride during induction. An alternate explanation, which as yet has density gradient. Growth in H20 followed by induction in 95% never been conclusively eliminated, is that induction involves D2O shifts the density of the enzyme to 1.3130 g/cm3, indicating de novo synthesis during induction. In the reciprocal experi- de novo synthesis of an "activator" protein, presumably en- ment, i.e., growth in 80% D20 followed by induction in either coded in the qa-l gene. This hypothesis states that the qa-2, qa-3, 95% D20 or H20, the densities of catabolic dehydroquinase and qa-4 polypeptides are synthesized constitutively and that were 1.3135 and 1.2800 g/cm3, respectively. Because growth on during induction the newly synthesized qa-1 gene product D20 does not affect the density of the H20-induced enzyme, serves to "activate" these polypeptides. The data reported there can be no significant synthesis of catabolic dehydroqui- herein demonstrate conclusively, using density labeling, that nase prior to induction. Similar results were obtained for a second qa enzyme, quinate dehydrogenase (quinate:NAD+ at least two of the qa enzymes are synthesized de novo during oxidoreductase, EC 1.1.1.24). Thus, induction of two qa enzymes induction. This conclusion strongly supports the hypothesis that involves de nQvo protein synthesis, not enzyme activation or the qa-1 locus encodes a regulatory protein that acts in a positive assembly. fashion to initiate the synthesis of the qa enzymes, and elimi- nates the possibility that induction involves enzyme activation In Neurospora crassa, the catabolism of quinic acid to acetate rather than enzyme synthesis. for use as a carbon source has been amply studied (1-3). Recent interest has centered on the initial three inducible catabolic MATERIALS AND METHODS enzymes necessary to convert quinic acid to protocatechuic acid (4-13). These enzymes are not physically associated with one Strains and Growth Conditions. Neurospora crassa qa-4 another, yet the three genes encoding them are tightly linked (strain M-18) and qa-3 (strain M-16) mutants, both derived from on chromosome VII near me-7. In this gene cluster, qa-2 en- wild-type strain 74-OR-23-LA (8), were used in this work. codes catabolic dehydroquinase (5-dehydroquinate dehydra- Mycelial cultures were grown from conidial inocula in 2000-ml encodes Erlenmeyer flasks containing 500 ml of Vogel's minimal salts tase; 5-dehydroquinate hydro-lyase, EC 4.2.1.10), qa-3 (13) and 2.0% sucrose (growth medium). The cultures were quinate dehydrogenase (quinate:NAD+ oxidoreductase, EC agitated on a rotary shaker at 300 rpm for 18 hr at 300. For 1.1.1.24), and qa-4 encodes 5-dehydroshikimate dehydratase. induction, the cultures were transferred, after exhaustive All three enzymes have now been purified to homogeneity by washing with sterile H20, to 2000-ml Erlenmeyer flasks con- quite different purification schemes. Catabolic dehydroquinase taining 500 ml of Vogel's minimal salts, 1% (vol/vol) glycerol, is a multimeric (homopolymeric) enzyme with a molecular and 0.3% quinic acid (induction medium). The flasks were weight of ca 220,000 composed of approximately 22 identical an additional 6 hr. At subunits (11). Quinate dehydrogenase has a molecular weight returned to the shaker and agitated for of ca 41,000 and is a monomer.* The third enzyme, dehydro- this time the pads were harvested, frozen at -70°, and lyoph- shikimate dehydrase, has recently been purified and it, too, is ilized. Slight modifications were made for growth and induction a monomer, with a molecular weight of ca 37,000 (P. Stroman, in the presence of D20. For growth of conidia to the mycelial W. R. Reinert, and N. H. Giles, unpublished). This qa gene stage, 400 ml of D20 were substituted for 400 ml of H20 in the a which growth medium to give a final concentration of 80% D20. cluster also includes fourth gene, qa-1, apparently Because the presence of D20 causes slower growth, the flasks regulates the transcription of the three other genes in the cluster. were allowed to shake for 24 hr at 300. For induction in D20, both H20-grown and D20-grown mycelia were first washed The costs of publication of this article were defrayed in part by the in flasks payment of page charges. This article must therefore be hereby marked with sterile H20 and then placed 500-ml Erlenmeyer "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * J. L. Barea and N. H. Giles, unpublished data. 4256 Downloaded by guest on September 30, 2021 Biochemistry: Reinert and Giles Proc. Nati. Acad. Sci. USA 74 (1977) 4257 containing 100 ml of the induction medium prepared in 95% D20. These flasks were shaken at 300 for 6 hr. Enzyme Assays. The assays employed for catabolic dehy- droquinase, quinate dehydrogenase, and dehydroshikimate dehydrase have been reported previously (14). Specific activities are expressed as nanomoles of product formed per min/mg of protein at 370. Enzyme Extraction. The lyophilized mycelia were ground in a Wiley mill and suspended at 20 g/ml in the extraction buffer. For extraction of catabolic dehydroquinase, Buffer A I- [100 mM potassium phosphate (pH 7.5)/0.4 mM dithiothrei- U..5F tol/0.1 mM ethylenediaminetetraacetic acid (EDTA)/0.1 mM C 4i phenylmethylsulfonyl fluoride/2.5 mM magnesium chloride] U was used and the enzyme was purified as described previously (11) through the ammonium sulfate precipitation stage. For quinate dehydrogenase, the extraction buffer was 20 mM po- tassium phosphate (pH 7.1) containing 1 mM EDTA, 1 mM a-thioglycerol, and 0.1 mM phenylmethylsulfonylfluoride. Dehydroshikimate dehydrase was extracted in Buffer A without EDTA. The first two steps of purification (initial extraction and RNase/DNase treatment) for both quinate dehydrogenase and I 2 3 4 5 6 I 2 3 4 3 6 dehydroshikimate dehydrase were identical to those for cata- TIME (HOURS) bolic dehydroquinase (11). Both enzymes were then subjected FIG. 1. Inhibition of enzyme induction with cycloheximide. The to ammonium sulfate fractionation, with the 40-50% fraction specific activities of (A) dehydroquinase (0) and (B) dehydroshiki- being retained for the dehydrogenase and the 0-40% fraction mate dehydrase (0) increase rapidly over a 6-hr period in the presence for the dehydrase. For all three enzymes the ammonium sulfate of an inducing medium. No increase in either enzyme's specific ac- precipitates were resuspended in 3.5 ml of their respective tivity (0) is observed when cycloheximide is added to the inducing extraction buffers and then dialyzed overnight against 100 media at zero time. Cycloheximide was also added after 3 hr of in- volumes of the same buffer to remove all traces of ammonium duction (c), and the culture was shaken for 3 more hr (x). sulfate. Any precipitated protein was removed by centrifuga- tion at 20,000 X g for 10 min. Inhibition with Cycloheximide. Nine 2000-ml Erlenmeyer RESULTS flasks containing 500 ml of growth medium were inoculated Inhibition of Induction with Cycloheximide. Noninduced with conidia from strain M-16 and allowed to grow for 18 hr mycelia contain a low yet measurable amount of both catabolic at 300. The mycelia from all the flasks were collected by fil- dehydroquinase and dehydroshikimate dehydrase, as seen in tration and placed in a 6000-ml Erlenmeyer flask containing Fig. 1 (the zero time points). Upon induction with quinic acid 2000 ml of sterile water and stirred for 1 hr to exhaust all the specific activities of both enzymes rise coordinately after available carbon sources. The mycelia were again collected by a short lag (approximately 30 min) as previously reported by filtration and the pad was cut into nine pieces (each about 10 Chaleff (9).
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