Saccharomyces fragilis

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1 JOURNAL OF BACTERIOLOGY, July 1973, p Copyright 1973 American Society for Microbiology Vol. 115, No. 1 Printed in U.S.A. Repression of In Vivo Synthesis of the Mitochondrial Elongation Factors T and G in Saccharomyces fragilis D. RICHTER' The Rockefeller University, New York, New York 11 Received for publication February 1973 In vivo synthesis of the mitochondrial elongation factors T and G in the yeast Saccharomyces fragilis can be repressed. Enzymatic activity assays and immunochemical titration methods reveal that cells grown in the presence of 8% glucose or in the absence of oxygen contain relatively lower amounts of mitochondrial elongation factors than cells grown in the presence of lactate. In contrast, in vivo production of the cytoplasmic elongation factors 1 and does not respond to such a change of extracellular conditions. The rate of growth does not affect the level of the mitochondrial elongation factors. Production of both enzymes is almost constant during logarithmic growth, but decreases when the stationary phase is reached. Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not block but, rather, seems to enhance the in vivo synthesis of mitochondrial T or G. In an in vitro system from yeast mitochondria, it was found that deoxyribonucleic acid (DNA) from the Escherichia coli phages T3 and T7 directed the synthesis of phage-specific enzymes, indicating that bacterial and mitochondrial transcription and translation processes are rather alike (17). These data also demonstrated that the protein-synthesizing machinery isolated from mitochondria was autonomous and fully capable of synthesizing specific enzymes in vitro. However, in vivo, the assemblage, regulation, and control of this system seem to depend strongly on events taking place in the nucleus and cytoplasm. The enzymes of the mitochondrial protein-synthesizing system studied so far have been found to be coded by nuclear, rather than mitochondrial, DNA. For instance, a yeast mutant deficient in mitochondrial DNA contains a complete set of the mitochondrial elongation factors T and G (EF-T and EF-G) (14, 16, ); EF-T is required to transfer aminoacyl-transfer ribonucleic acid (trna) to the ribosomes; EF-G catalyzes the translocation step (1). This yeast mutant also contains the mitochondrial transformylase, an enzyme that catalyzes the formylation of the initiator Met-tRNA (D. Richter, unpublished dat. Studies with antibiotics that specifically inhibit mitochondrial protein synthesis have revealed that ribosomal proteins from mitochondria of Neurospora crassa are synthesized on cytoplasmic ribosomes (7, 1, 13). More recently, it has been reported that nuclear DNA directs the synthesis of the mitochondrial RNA polymerase (1). All these data imply a regulation and control of the mitochondrial proteinsynthesizing machinery by nuclear DNA; however, to date, nothing is known about how the flow of information between nucleus and mitochondria is mediated. In addition, in yeast, the mitochondrial machinery may also respond to extracellular factors such as oxygen supply or carbon source (19). The purpose of this study was to see whether these extracellular conditions influence the synthesis of the enzymes participating in the mitochondrial protein synthesizing system. I chose as the particular target the product of mitochondrial EF-T and EF-G in the yeast Saccharomyces fragilis. The data show that their synthesis was repressed in the presence of high concentrations of glucose or under anaerobic conditions. No repression of synthesis of the cytoplasmic elongation factors 1 or (EF-1 and EF-, the former being equivalent to EF-T, the latter to EF-G) was observed. When mitochondrial protein synthesis was blocked by addition of chloramphenicol to the ' Present address: Max-Planck-Institut fur Molekulare culture, the synthesis of EF-T and EF-G was Genetik, Ihnestrasse 63/73, 1 Berlin 33, Germany. stimulated rather than inhibited. 5

2 VOL. 115, 1973 REPRESSION OF MITOCHONDRIAL T AND G 53 MATERIALS AND METHODS Growth conditions. The growth conditions of S. fragilis were essentially the same as those previously described (16, 1, ). When cells were grown in the presence of chloramphenicol, 4 mg of the antibiotic were added, and the medium contained.5% glucose (16). Anaerobic conditions were obtained by the method of Criddle and Schatz (3) by passing purified nitrogen through the medium which contained.5% glucose (16). Cells were harvested from the midlogarithmic phase. Preparation of the high-speed supernatant fraction (S-1). When necessary, mitochondrial and cytoplasmic elongation factors were separated as described (18). 7S ribosomes were prepared from E. coli (4); 8S ribosomes were obtained from yeast cytoplasm (15) or reticulocyte cells (6). To determine the relative amounts of mitochondrial elongation factors, cells were harvested and suspended in vol of buffer A [mm tris(hydroxymethyl)aminomethanehydrochloride (ph 7.4), 1 mm dithiothreitol ]. The cell suspension was homogenized in a French pressure cell at 1, lb/in, and then centrifuged at 1, x g for 1 min. The cell pellet was resuspended in two volumes of buffer A and rehomogenized. As shown recently (18), this treatment causes not only disruption of the cell wall, but also of the mitochondrial particles, and consequently these homogenates contained both sets of elongation factors, cytoplasmic as well as mitochondrial. Since the cytoplasmic factors did not interact with 7S ribosomes from E. coli or mitochondria, their presence in the supernatant fraction did not affect the assay for mitochondrial EF-T or EF-G (6, 15, 18). The cell extract was centrifuged at 18, x g for min, and the supernatant fraction was carefully collected and recentrifuged at 15, x g for h. To determine the level of mitochondrial EF-T and EF-G, this S-1 fraction, rather than isolated mitochondria, was used. Leakage of proteins from mitochondria during the isolation procedure may vary with different growth conditions (9) and therefore could falsify the experiments. Determination of mitochondrial EF-T and EF-G in the S-1 fraction. For the enzymatic activity assay, the specific activity of mitochondrial EF-T or EF-G was measured by polyphenylalanine synthesis in the presence of the complementary purified mitochondrial elongation factor and 7S ribosomes from E. coli. The specific activities of both enzymes were obtained from experiments with a linear dependence on either EF-T or EF-G concentration (16). Similar experiments were carried out with the cytoplasmic EF-1 and EF- by using 8S ribosomes from yeast or reticulocyte cells. The immunochemical assay was the method outlined by Gordon (5). A constant volume of antiserum prepared against purified EF-T or EF-G (16) was titrated with increasing concentrations of the elongation factors. Antibody-antigen formation was determined at AS8 nm at 5 C after 1 min. The turbidity obtained at A8* nm was related to milligrams of S-1 protein and was derived from experiments with a linear dependence on either EF-T or EF-G concentration. Thus, the A8. units reflect the relative amount of mitochondrial elongation factors present in the cell. RESULTS Synthesis of mitochondrial EF-T and EF-G in cells grown in the presence of different carbon sources. High glucose concentrations repress mitochondrial activity in yeast cells, but in the presence of lactate or low concentrations of glucose repression is released (9, 19). To determine whether the relative amount of mitochondrial elongation factors in the cell was affected by the carbon source, S. fragilis was grown in media containing either % lactate,.5% glucose, or 8% glucose; the cells were analyzed for mitochondrial EF-T or EF-G at different time periods. The specific activities of both enzymes were much lower in cells cultured with 8% glucose (Fig. 1C) as compared to lactate-grown cells (Fig. 1A); with.5% glucose in the medium, synthesis of both factors was less repressed (Fig. 1B). In all experiments, the specific activity of mitochondrial EF-T was slightly less than that of its counterpart, EF-G, which could be explained by the instability of the former. In addition, Fig. 1 indicates that during the logarithmic phase the rate of growth did not affect the synthesis of both mitochondrial enzymes. In fact, yeast cells with slightly longer generation times than lactate-grown cells showed rather high specific activities for mitochondrial EF-T and EF-G. This is in contrast to the in vivo synthesis of elongation factors in E. coli where the highest production was obtained in cells with short generation times (5). The in vivo production of mitochondrial elongation factors decreased when the cells reached the stationary phase (Fig. 1). Although EF-T and EF-G were coded on nuclear DNA and synthesized on 8S ribosomes of the cytoplasm (14, 16, ), their production appeared to be repressed by high glucose concentrations. This conclusion remained tentative; since the calculation of their production was based on the polyphenylalanine assay, the possibility that extracts from cells grown in glucose contained unspecific inhibitors could not be excluded. Unless these inhibitors were rather specific for the mitochondrial system, cytoplasmic protein synthesis should also be affected. Similar calculations for the cytoplasmic EF-1 and EF-, however, revealed that the specific activities of both enzymes did not vary with different growth conditions or carbon source (not shown). In addition, S-1 protein from glucose-grown cells did not inhibit the activity of mitochondrial EF-T or EF-G isolated from lactate-grown cells (not shown), strengthening the point that synthesis of the two en-

3 54 RICHTER r'j E a} ': Coo o,w (1_ C o E ) 4 3 D >/EF~~E-G % lactate I 3.5% glucose wef-t (B) 33 [ (C) 8% glucose 4 (A) 3- II- EF-G Time of growth (hr) FIG. 1. In vivo synthesis of mitochondrial EF-T and EF-G under different growth conditions. S. fragilis was grown as described (16, 1, ) in 15-liter flasks of a New Brunswick fermentor at 37 C with stirring (8 rpm) and aeration ( liters of air/min). Samples of.5 liter were harvested at the times indicated and processed as described in Materials and Methods. The arrows indicate the time when the stationary phase of the culture was reached. zymes could be repressed under appropriate conditions. So far, the validity of our calculations for the repression of synthesis of both enzymes has been based on the measurement of their specific activities. Recently, Gordon (5) and Leder et al. (8) studied the regulation of in vivo synthesis of bacterial elongation factors by using, among other assays, an immunochemical method. A similar approach was made in the following experiments. As reported (16), antibodies prepared against mitochondrial EF-T or EF-G were highly specific for either factor and showed no cross-interaction. To determine the relative amounts of both factors in the S-1 preparations, they were titrated with anti-t or anti-g serum; the antibody-antigen formation was measured at A38 nm. The experiments shown in Table 1 confirm the results obtained with the polymerization assay. The S-1 fraction from J. BACTERIOL. cells grown in glucose contained significantly less A8., units than those grown in lactate. Production of mitochondrial EF-T and EF-G in aerobically and anaerobically grown celis. The data described above revealed that conditions that repress mitochondrial activity also affect synthesis of mitochondrial EF-T and EF-G. Hence, anaerobic growth conditions should also repress their production. The specific activities of both factors were almost 5% lower in the anaerobically grown cells than in the control experiments where aeration was optimal (Table ). The less pronounced repression of mitochondrial EF-T and EF-G synthesis may be explained by the difficulty in maintaining complete anaerobic conditions during growth and harvesting of the cells. Synthesis of the cytoplasmic EF-1 and EF- was not affected under anaerobiosis. Effect of chloramphenicol. Chloramphenicol inhibits the synthesis of mitochondrial proteins such as those of the inner membrane or some of the subunits of the cytochrome oxidase or of the oligomycin-sensitive adenosine triphosphatase (, 9, 19). Thus, mitochondrial activity resembles that of repressed cells. It was TABLE 1. Immunochemical assay of mitochondrial EF-T and EF-G AFon units of S-1 fraction from cells mitochondriaa grown in: EF-T EF-G.% Lactate % Glucose % Glucose afor definition Ac5O of units, see Materials and Methods. TABLE. In vivo synthesis of mitochondrial and cytoplasmic elongation factors in the presence and absence of oxygena Growhcnditons Phenylalanine polymerized (pmol per mg of S-1 protein) Mitochondrial Cytoplasmic EF-T EF-G EF-1 EF- Aerobic Anaerobic Forowthe... conditions s1.n a Polyphenylalanine synthesis was measured at 37 C for 1 min by using 5,ug of E. coli ribosomes or 5,ug of cytoplasmic ribosomes from reticulocyte cells. For other assay conditions see references 16 and 17. Protein concentration was measured by the method of Lowry et al. (11).

4 VOL. 115, 1973 REPRESSION OF MITOCHONDRIAL T AND G 55 of interest to see whether this antibiotic could also affect the level of mitochondrial EF-T and EF-G in the cell. Again, the amounts of both enzymes were assayed in terms of activities. In contrast to the metabolically repressed cells, inhibition of mitochondrial protein synthesis by chloramphenicol did not cause inhibition of the synthesis of the two elongation factors (Table 3). Their relative amounts were rather higher in cells treated with chloramphenicol than in the control. These results clearly indicate that inhibition of mitochondrial protein synthesis did not parallel the glucose-linked repression of production of mitochondrial EF-T or EF-G. In light of the recent findings (14, 16, ), this is not unexpected since both factors are products of the chloramphenicol-insensitive system of the cytoplasm. DISCUSSION In vivo synthesis of mitochondrial EF-T and EF-G could be repressed in the presence of high glucose concentrations or in the absence of oxygen. A number of controls exclude that this repression was due to our assay conditions. From the experiments with chloramphenicol (Table 3) and from previous findings (14, 16, ) with mutants lacking mitochondrial DNA, the mitochondrial elongation factors are entirely products of a nuclear gene, and therefore this repression cannot be attributed to the absence of an active mitochondrial system. The data reported here imply, rather, that the expression of nuclear genes specifying mitochondrial elongation factors is under an additional control, presumably catabolic regulation. This control seems to be specific since production of EF-1 and EF- was not repressed. In this respect, it is of particular interest that in cells of Euglena gracilis the in vivo synthesis of elongation factor T, which is specific for 7S ribosomes, was increased on the induction by light of cells TABLE 3. In vivo synthesis of mitochondrial EF-T and EF-G in cells treated with chloramphenicola Growth conditions Phenylalanine polymerized (pmol S-1 per mg of protein) EF-T EF-G Without chloramphenicol With chloramphenicol a Polyphenylalanine synthesis was measured at 37 C for 1 min by using 5 ug of E. coli ribosomes per 15 gliters of total assay volume. grown in the dark, whereas no increase was observed for its cytoplasmic counterpart EF-1 (. Ciferri, personal communication). In conclusion, the data show that when synthesis of mitochondrial enzymes is not required, the cell economically preserves their production by responding to extracellular conditions. It remains to be seen how this response is mediated and how the mediator affects the expression of those nuclear gene products that function in mitochondrial particles. ACKNOWLEDGMENTS The work reported here was supported by Public Health Service grant GM-1397 from the National Institute of General Medical Sciences, to Fritz Lipmann, whose continued encouragement and helpful discussions are gratefully acknowledged. LITERATURE CITED 1. Barak, Z., and H. Kuntzel Induction of mitochondrial RNA polymerase in Neurospora crassa. Nature N. Biol. 4: Borst, P Mitochondrial nucleic acids. Annu. Rev. Biochem. 41: Criddle, R. S., and G. Schatz Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties. Biochemistry 8: Gordon, J Hydrolysis of guanosine 5'-triphosphate associated with binding of aminoacyl-trna to ribosomes. J. Biol. Chem. 44: Gordon, J The regulation of the in vivo synthesis of the polypeptide chain elongation factors in Escherichia coli. 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