Inhibition of the import of mitochondrial proteins by RNase.

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May 15, 2015 - described in the legends to Figs. 1 and 2. RNase was pretreated by boiling for 10 min prior to use, in order to remove DNase contami- nation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 14,Issue of May 15, pp. 6153-6155,1986 0 1986 by The American Society of Biolog.lcal Chemists Inc. Printed in d S A .

Communication Inhibition of the Import of Mitochondrial Proteins by RNase* (Received for publication, December 12,1985)

David Burns andAlfred LewinS From the Department of Chemistry and Instituteof Molecular Biology, Indiana University, Bloomington, Indiana 47405

tion system. Nevertheless, we do not feel that these experiments demonstrate an RNA requirement for import. In our system, inhibition is dependent on the presence of ribosomes; yet import of mitochondrial proteins occurs efficiently if ribosomes are first removed from the cell-free translation system. In addition, substantial inhibition of import can be achieved by incubating the translation mixture with other proteins prior to import. EXPERIMENTAL PROCEDURES

RNase treatment of a cell-free translation system preventstransport of mitochondrial precursor proteinsMaterial~-[~S]methionine(>lo00 Ci/mmol) was obtained from Amersham or New England Nuclear. Dehydrated culture medium from that system into isolated yeast mitochondria. This from Difco. Enzymes and fine chemicals were from Boehringer inhibition dependson the presence of ribosomes in thewas Mannheim, Calbiochem, or Sigma. Porcine insulin was a generous reticulocyte lysate; if they are cleared by centrifuga- gift of Dr. Ruth Gurd (Departmentof Chemistry, Indiana University). tion, RNase treatment does not specificallyinhibit proCell-free Protein Synthesis and Protein Import-Translation of tein uptake by mitochondria.Since protein import can yeast mRNA in a reticulocyte lysate system pretreated with Staphyoccur in the absence of polyribosomes, RNase treatlococcus aureus nuclease (14) and mitochondrial isolation were perment does not degrade a structure essential for this formed by our previously reported procedure (15). Import of proteins process.Rather,theinhibition maybean effect of into mitochondria was also performed as described in Ref. 15, except gel filtration of the lysate on Sephadex G-25 was omitted. degraded ribosomes.

Immunological Techniques-Antibodies were prepared in rabbits by standard protocols and were used as whole sera. Immunoprecipitation using glutaraldehyde-fixed S. aureus cells was done as described in Ref. 16. Following washing of S. aureus cells, proteins were Most mitochondrial proteins are synthesized in the cyto- eluted in electrophoresis sample buffer and separated on sodium plasm. As with many proteins which must be transported dodecyl sulfate-polyacrylamide gels (17). Protein bands from immuthrough membranes, mitochondrial proteins areusually made noprecipitates were detected by fluorography. RNase Pretreatment of Reticulocyte Lysate-Following translation as larger precursors which have N-terminal amino acid extensions not found in the mature proteins (1, 2). Unlike the of yeast messenger RNA, polyribosomes were sometimes pelleted by centrifugation at 139,000 X g a t 4 “C for 30 min. Supernatants or vectorial translation of secreted proteins, mitochondrial pre- lysates not subjected to centrifugation were then treated with RNase cursors are usually transported after their synthesis on free (Sigma type I11 A) or with other proteins at either 27 or 37 “C as polyribosomes is complete, although there is evidence of co- described in the legends to Figs. 1 and 2. RNase was pretreated by translational importof certain mitochondrial proteins in yeast boiling for 10 min prior to use, in order to remove DNase contami(3-5). Binding of cytosolic precursors of mitochondrial pro- nation. To inactivate RNase, the enzyme was treated with 1.4 mM teins to mitochondria is thought to involve protein receptors diethyl pyrocarbonate for 12 h. Subsequently, Tris-HC1 (pH 7.4) was added to 20 mM and thesolution was boiled to remove residual diethyl on the surface of the organelle. In addition, a cytosolic factor pyrocarbonate and itsbreakdown products. RNase activity was monis required for the transport of yeast (6) and mammalian itored by treating high M, yeast RNA with active or inactive RNase proteins (7, 8) into isolated mitochondria. fractions and examining the integrity of the RNA by denaturing gel Recently RNase treatment was used to inhibit the import electrophoresis (18). Other Methods-Quantitation of immunoprecipitated mitochonof precursor to ornithinetranscarbamylase from arabbit reticulocyte lysate translation system into ratliver mitochon- drial proteinswas done using a Joyce-Loebl microdensitometer. Several exposures of autoradiograms were scanned to assure that darkdria (9). Those studies (9) also demonstrated that precursor ened bands had not saturated the x-ray film.

to ornithine transcarbamylase is present in ahigh molecular weight fraction of the translation system. The authors concluded that theprecursor is complexed with an RNA-containing component essential for import. Because an RNA-containing signal recognition particle isrequired for translocation of secretory proteins (10-13), such a particle is plausible for mitochondrial protein translocation, even though there is no formal requirement for translational arrest. We have duplicated the observations concerning the RNase sensitivity of import for precursors of two yeast (Succharomyces cereuisiae)mitochondrial proteins: the P subunit of F1ATPase and citrate synthase. We have also detected these precursors as part of a large complex in the cell-free transla-

*This research was supported by United States Public Health Service Grant 2R01 GM29387-04A1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Fellow of the Institutefor Molecular Biology which is supported by the Indiana Corporation for Science and Technology.

RESULTS AND DISCUSSION

RNase Inhibits the Transport of Precursors into Mitochondria-To assess the effect of RNase treatment, we studied the import of two proteins to which we have strong and specific antisera, the ,f3 subunit of F1-ATPase and citrate synthase. Both proteins are localized in the matrix space and consequently must cross both mitochondrial membranes after synthesis on cytoplasmic ribosomes. Since our results were identical for both proteins, we show only the data obtained for FIB. Following translation of yeast mRNA ina reticulocyte lysate protein synthesis system, the translation mixture was treated for 10 min with a high level (100 pg/ml) of ribonuclease A. The level and the commercial source of the enzyme were identical to those specified by Firgaira et al. (9). To assure that contaminating DNase had been inactivated, the enzyme had been immersed in aboiling water bath for 10 min and then reassayed for RNase activity by digestion of yeast

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Import of Proteins Mitochondrial

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RNA. After RNase pretreatment, the import of mitochondrial precursors from the lysate into freshly isolated yeast mitochondria was assayed under our standard import conditions (15). Import of precursor protein is determined by the level of mature (processed) protein associated with the mitochondrial pellet (Fig. L4, lane 1; Fig. lB, lane 1 ). Inhibition is the per cent reduction in import relative to our regular import conditions with the same mitochondria and lysate. When translation reactions were treated with RNase at

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37 "C immediately following protein synthesis, importof precursor to the p subunit (pre p) was inhibited significantly (96%) (Fig. lA, lane 2 ) . Inhibition was less when lysates were treated with RNase a t 27 "C (45% inhibition) (Fig. lA, lane 3 ) . Incubation for the same duration without RNase caused some reduction of import relative to our regular import reaction (Fig. lA, lanes 4 and 5 ) . No RNase Inhibition afterHigh-speed Centrifugation-Our usual import procedure involves removal of ribosomes from the reticulocyte lysate by centrifugation. When lysates were subjected to centrifugationa t 139,000 x g for 30min toremove ribosomes prior to RNase, little inhibition of import by RNase was observed (Fig. lB, lanes 2 and 3 ) .In this instance, import was not significantly different than when lysates were preincubated without RNase(Fig. lB, lanes 4 and 5 ) . Therefore, it appearsthatRNaseinhibition of importdependsonthe presence of reticulocyte ribosomes in the reaction. To determine if a component of the ribosomal pellet was necessary for the RNase inhibitionof import, we added ribosomes back to a translation mixture from which they had been cleared by centrifugation. The crude ribosomal pellet was rinsed and then resuspended in import reaction buffer and added to the lysate before RNase treatment. Restoring the ribosomes also restored the inhibition of import by RNase as described above. Adding back ribosomes without RNase treatment had noeffect on import. T o determine whether the RNase effect was specific, we treated lysates before the import reaction with inactivated RNase as well as with other proteins at thesame concentration (100 pg/ml) (Fig. 2). While RNase reduced the import of FIB into mitochondria by 46%, the same amount of RNase pretreated with diethyl pyrocarbonate caused no reduction of 100 pg/ml of DNase I (Boehringer import. On the other hand, Mannheim), lysozyme (Sigma, TypeI),or porcine insulin (Calbiochem) each reduced import by 25-30%. When tested with yeast RNA, the DNase, lysozyme, and insulin preparations we used showed no significant RNase activity. Consequently, their inhibition of import did not reflect a contaminatingRNase activity.Bovine serumalbumin showed no

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FIG. 1. Ribonuclease inhibition of import depends on the presence of ribosomes. Yeast mRNA was translated in a rabbit reticulocyte lysate containing [35S]methionine.After 60 min, half of the reaction mixture was subjected to centrifugation a t 139,000 X g for 30 min to remove ribosomes. Lysates with or without ribosomes were incubated a t 27 or 37 "C for 10 min in the presence or absence of heat-treated ribonuclease A (100pglml). The lysate fractions (80 PI) were then mixed with freshly isolated mitochondria(110pg). After 40 min at 30 "C, mitochondria were separated from the incubation mixture by centrifugation. The mitochondrial pellet was washed by resuspension and centrifugation, and both pellet and supernatant fractions were dissociated withsodium dodecyl sulfate at 95 "C. Samples were then diluted with abuffer containing TritonX-100 (50 mM Tris-C1, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% (w/v) Triton X-100)and prepared for immunoprecipitation. Immunoprecipitates of Flp subunit from mitochondria and post-mitochondrial supernatants following import were separated on 10%sodium dodecyl sulfatepolyacrylamide gels. The figure is a fluorogram of two such gels. A, import of p subunit in the presence of ribosomes. In each lane S means post-mitochondrial supernatant and M means mitochondrial pellet. Lane 1 , standard import reaction; lane 2, RNase treatment at 37 "C; lane 3, RNase treatment a t 27 "C; lane 4, mock pretreatment 37 "C; lane 5,mock pretreatment 27 'C. B, import of the subunit in the absence of ribosomes. Lanes were loaded in the same order as gel A.

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FIG.2. Active RNase is required for inhibition of import. Lysates were preincubated with ribonuclease A and other proteinsa t the same concentration (100 pglml) at 37 "C and then assayed for FIB import. This bar graph is derived from densitometer tracings of a fluorogram, similar to those shown in Fig. 1. The per cent import in each trial was first determined by dividing the amount of mature FIBfound with the mitochondrial pelletby the total amount of mature and precursor Flp associated with both pellet and supernatant fractions. Inhibition is the per cent reduction in import relative to our regular import conditions with the same mitochondria and lysate. Mock, mock preincubation of lysate; RNase, addition of ribonuclease A; DEPC, addition of ribonuclease A pretreated with diethyl pyrocarbonate. BSA, bovine serum albumin. DNase, deoxyribonuclease I.

Import of Mitochondrial Proteins effect on import. We conclude that the inhibitory effect of RNase on import is attributablea t least in part to itsnucleolytic activity but that other proteins (DNase, lysozyme, and insulin) canalso reduce protein uptakewhen added in similar concentrations. Ribosomes Are Not Required for Import-Since RNase treatment reduced protein import only in the presence of ribosomes, we wondered if removing the polysomes by centrifugation reduces the efficiency of protein translocation into mitochondria. In our hands import was as efficient after highspeed centrifugation of the reticulocyte lysate as it was before removal of the ribosomes by this step. In three separate experiments, import of p subunit in the absence of polysomes averaged 87.9% while import in the presence of polysomes averaged 81.5%. (The difference is not statistically significant.) In addition, the rateof import of mitochondrial proteins was the same in the presence or absence of polysomes in the import incubation (Fig. 3). The centrifugation step may not remove all monosomes or ribosomal subunits from the lysate. Conversely, other large components besides ribosomes may be sedimented. Nevertheless, the centrifuged lysate, which is RNase-insensitive, is competent to import mitochondrial proteins in uitro. Most other laboratories studying the transport of proteins into isolated mitochondria routinely pellet cytoplasmic ribosomes prior to theimport reaction (7,8, 16). The Requirement for a Signal Recognition Particle-The transit of mitochondrial protein precursors from the cytoplasm does not require arrest of translation, because synthesis of precursors is usually completed before membrane translocation begins. The translation arrest and protein transport activities of the signal recognition particle aredissociable (19, ZO), however, and Firgaira et al. (9) observe that RNase inhibits protein import into mitochondria and that the precursor to ornithine transcarbamylase is present in a highmolecular weight fraction of the reticulocyte lysate. They infer that an RNA-containing particle mediates import of

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proteins from the cytoplasm into mitochondrion. We confirm and extend theirobservations for precursors of yeast mitochondrial proteins. We have controlled for the effect of DNase contamination by boiling the RNase, we have checked for inhibition by other proteinsat similar concentrations; and we have monitored the effect of inactivated RNase on mitochondrial protein import. Active RNase inhibits import a t 37 "C, but some inhibition is attributable to the incubation alone. Nevertheless, we do not conclude that an RNA component is essential for import. If ribosomes are cleared from the cell-free system prior to RNase treatment, little or no inhibition of import is detected. Since lysate ribosomes are not required for efficient importation of proteins into mitochondria, there is no evidence for an RNA essential for import. The observation of precursors in a highmolecular weight fraction (9) may be caused by aggregation of newly made proteins rather than by interaction of precursors with a lysate component. We have detected substantial aggregation of mitochondrial precursors with other translation products when lysates are incubated at 37 "C following translation (data not shown). The most likely effect of RNase in our experiments and, by analogy, in those of Firgaira et al. (9) is to degrade ribosomes. This breakdown may inhibit import either bycomplexing with precursors or by interfering with the mitochondria. If mitochondria are uncoupled by degradation products, import of proteins will be blocked.The essential pointis that RNase treatment of centrifuged lysate did not reduce import compared to mock incubation at 37 "C. Import of proteins into mitochondria may well require an RNA-containing particle from the cytoplasm. At present, however, there is no convincing evidence to demonstrate this requirement. Ackmwledgmnts-We would like to thank Peggy Ericson for preparing the manuscript and Dr. John Richardson for his expert advice. REFERENCES 1. Schatz, G., and Butow, R. A. (1983) Cell 32, 316-318

2. Hay, R., Bohni, P., and Gasser, S. (1984) Biochim. Biophys.Acta 779,65-87 3. Kellems, R.E., and Butow, R. A. (1972) J. Biol. Chem. 247, 8043-8050 4. Ades, I. Z., and Butow, R. A. (1980) J. Biol. Chem. 255, 99189924 5. Suissa, M., and Schatz, G. (1982) J. Biol.Chem. 257, 1304813055 6. Ohta, S., and Schatz, G. (1984) EMBO J. 3,651-657 7. Argan, C., Lusty, C. J., and Shore, G. C. (1983) J. Biol. Chem. 258,6667-6670 8. Miura, S., Mori, M., andTatibana, M. (1983) J. Biol. Chem. 258, 6671-6674 9. Firgaira, F. A., Hendrick, J. P., Kalousek, F., Kraus, J. P., and Rosenberg, L. E. (1984) Science 226, 1319-1322 10. Walter, P., Ibrahami, I., and Blobel, G. (1981) J. Cell Biol. 91, 454-550 11. Walter, P., and Blobel, G . (1981) J. Cell Biol. 91, 551-556 10 20 30 40 50 60 12. Walter, P., and Blobel, G. (1981) J. Cell Biol. 91, 557-561 13. Walter, P., and Blobel, G. (1982) Nature 299, 691-698 TIME (MINUTES) 14. Pelham, H. R. B., and Jackson, R. J. (1976) Eur. J. Biochem. 67, FIG. 3. The rate of mitochondrial protein import is the same 247-265 in the presence or absence of ribosomes. Two 1.5-ml import 15. Lewin, A. S., and Norman, D. K.(1983) J. Biol.Chem. 258, reactions were prepared, one containing centrifuged lysate (139,000 6750-6755 X g) and one containinguncentrifuged lysate. Freshly prepared yeast 16. Gasser, S. M.,Daum, G., and Schatz, G. (1982) J. Biol.Chem. mitochondria were mixed into both reactions at 30 "C. At various 257, 13034-13041 time points, a 200-pl aliquot from each reaction was removed, and 17. Douglas, M. G., Finkelstein, D., and Butow, R. (1979) Methods the import reaction was stopped by addition of EDTA to 1 mM, Enzyml. 56,58-65 immediate separation of the mitochondrial pellets from the superna- 18. Locker, J. (1979) Anal. Biochem.98,358-367 tant fractions, and chilling on ice. FIB import was assayed as detailed 19. Siegel, V., and Walter, P. (1985) J. Cell Biol. 100, 1913-1921 in Fig. 1. o " 0 , uncentrifuged lysate; U , centrifuged lysate. 20. Meyer, D. I. (1985) EMBO J. 4,2031-2033

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