©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Import of the Cytochrome Oxidase Subunit Va Precursor into Yeast Mitochondria Is Mediated by the Outer Membrane Receptor Mas20p (*)

Trevor Lithgow , Gottfried Schatz (§)

From the (1)Biozentrum, University of Basel, CH-4056 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Post-translational import of precursor proteins into yeast mitochondria is mediated by at least four protease-sensitive outer membrane proteins: Mas20p, Mas22p, Mas37p, and Mas70p. These ``import receptors'' recognize either the N-terminal targeting signal or some other feature of mitochondrial precursor proteins. The only exception to this general rule appeared to be the precursor to subunit Va of cytochrome c oxidase (COXVa). Although this precursor carries a typical N-terminal mitochondrial targeting sequence, its import into mitochondria has been suggested to be independent of the known import receptors. Here we show that if import into isolated yeast mitochondria is assayed under conditions in which binding of the COXVa precursor to mitochondria is rate-limiting, import is strongly inhibited by protease pretreatment of the mitochondria or by antibodies against Mas20p. Post-translational import of the COXVa precursor can thus proceed by the general, receptor-mediated pathway.


INTRODUCTION

One of the first steps of protein import into mitochondria is the binding of precursor proteins to several ``import receptor'' subunits on the cytosolic surface of the outer membrane(1, 2) . There is currently no direct assay for this step. Instead, binding to import receptors was initially inferred from the facts that protein import was inhibited by gentle protease treatment of intact mitochondria or by antibodies against surface-exposed outer membrane proteins(3, 4, 5, 6, 7, 8, 9) . In more recent assays, precursors prebound to deenergized mitochondria were chased into mitochondria upon reenergization of the organelles; this ``productive binding'' was sensitive to the treatments mentioned above(9, 10, 11, 12) . However, these measurements are only valid if the receptor-mediated step is rate-limiting for the import process. If this is not the case, inhibition or activation of surface receptors may not inhibit precursor import, since removal or inactivation of import receptors is rarely quantitative and since the different receptor subunits may partially substitute for each other(6, 8, 9, 13, 14) .

COXVa()is made as a precursor with a typical N-terminal basic, amphipathic targeting sequence composed of 20 amino acids(15) . Import of the precursor into the mitochondrial inner membrane uses the translocation channels of both mitochondrial membranes (16) and the translocase function of mhsp70 (17) but has been reported to bypass the usual import receptors on the mitochondrial surface. This receptor independence was inferred from the observations that import of the COXVa precursor was not inhibited by trypsin pretreatment of mitochondria (16) or by genetically deleting the Mas70p or Mas20p receptor subunits(17) . However, the published evidence suggested to us that these import assays had been performed in the presence of a functional excess of mitochondria and that binding of the precursor to mitochondria might not have been rate-limiting.

We have now reinvestigated this problem under assay conditions in which encounter of precursors with the mitochondrial surface is rate-limiting for import. Such conditions are mandatory for assessing the action of receptors in facilitating import of different precursor proteins. Our results show clearly that import of the COXVa precursor is mediated by trypsin-sensitive receptors, including Mas20p. Post-translational import of the COXVa precursor into isolated yeast mitochondria can thus occur by the usual, receptor-mediated import pathway.


EXPERIMENTAL PROCEDURES

Trypsin Pretreatment of Mitochondria

Mitochondria from the yeast Saccharomyces cerevisiae (strain D-273-10B, ATCC 25657) were isolated, their amount was quantified spectroscopically at 280 nm in the presence of SDS, and they were stored frozen as described previously(18) . In the standard treatment, mitochondria (0.5 mg) were thawed, diluted into a final volume of 0.5 ml of import buffer (20 mM HEPES-KOH, pH 7.4, 0.5 mM EDTA, 5 mM MgCl, 0.6 M sorbitol, 2 mM KHPO, 25 mM KCl) containing 50 µg of trypsin, and incubated for 30 min on ice. Import buffer (1.0 ml) containing 1.0 mg/ml trypsin inhibitor was then added to trypsin-treated or untreated mitochondria. After a further 5 min of incubation, the mitochondria were isolated by centrifugation, washed once in import buffer containing 0.1 mg/ml trypsin inhibitor and 1.0 mg/ml BSA, and finally resuspended in import buffer containing 1.0 mg/ml BSA.

In Vitro Import Assays

Trypsin-treated or untreated mitochondria were diluted as indicated into 3.0 ml of import buffer containing 1 mg/ml BSA, 0.5 mM ATP, and 0.5 mM NADH and incubated at the indicated assay temperature for 3 min. Reticulocyte lysate containing the S-labeled precursor was added, and at the indicated times, 0.5-ml samples were withdrawn to microcentrifuge tubes containing 60 µl of a termination mix (30 µg of carrier mitochondria in import buffer containing 100 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Proteinase K (4 µg) was then added, and after incubation for 20 min on ice, phenylmethylsulfonyl fluoride was added to 0.8 mM. The mitochondria were isolated by centrifugation and solubilized in SDS-sample buffer for electrophoretic analysis. Pretreatment of mitochondria with IgGs specific for the major outer membrane protein porin or for the receptor subunits Mas20p/Mas22p and Mas70p was as described(6) .

Miscellaneous

The SalI-EcoRI DNA fragment encoding the COXVa precursor protein was subcloned from plasmid pDpT7-5a (16) into pSP64 (Promega) for transcription with the SP6 RNA polymerase. SDS-PAGE was run on Tris-Tricine-buffered gels (19).


RESULTS AND DISCUSSION

The authentic precursors to F and HSP60 as well as the chimeric precursor SU9-DHFR are imported into yeast mitochondria via protease-sensitive receptors on the mitochondrial surface(6, 10, 12, 20, 21) . Import is not absolutely dependent on these receptors but is slowed 5-10-fold if these receptors are removed or inactivated. Thus, it is only possible to observe the influence of receptors on protein import if binding of the precursor to the mitochondrial surface is rate-limiting for the overall import process.

Two reports have suggested that import of the COXVa precursor into yeast mitochondria does not occur via the receptor-mediated pathway (16, 17). These import assays used mitochondrial concentrations equivalent to 0.5 mg/ml protein. We readily confirmed these findings; under these assay conditions, only the import of F was strongly inhibited by trypsin pretreatment of mitochondria (Fig. 1); import of SU9-DHFR was less affected and that of HSP60 or COXVa was completely unaffected.


Figure 1: At 0.5 mg/ml mitochondria in the import assay, trypsin pretreatment inhibits the import of only some mitochondrial precursors. Mitochondria were pretreated with trypsin (+) or left untreated (-) and then incubated at a final concentration of 0.5 mg/ml mitochondrial protein with S-labeled precursors to F (5 min at 25 °C), COXVa (5 min at 13 °C), HSP60 (5 min at 10 °C), or SU9-DHFR (5 min at 25 °C). Import was measured by removing external precursor with proteinase K and analyzing the mitochondria by SDS-PAGE, fluorography of the dried gels, and quantification of the fluorograms by PhosphorImager analysis. Photographs of the fluorograms are shown, together with the quantitation expressed as a percentage of import into mitochondria not treated with trypsin.



However, when we necessarily lowered the amount of mitochondria in the in vitro assay and coimported the precursors of F and COXVa, only the import of F was limited by the amount of mitochondria that had been used in the assay referred to above; at 25 °C, the amount of F imported increased linearly with the concentration of mitochondria in the assay at least up to 1.0 mg/ml (Fig. 2). In contrast, import of COXVa was only dependent on the concentration of mitochondria if that concentration was decreased about 20-fold and if the assay temperature was lowered to 13 °C.


Figure 2: Mitochondria limit the import rate of COXVa only at lower concentrations and temperature. A mixture of the precursors to F and COXVa was incubated with the indicated amount of mitochondria for 5 min at 25 °C; import of the precursor to COXVa was also tested for 5 min at 13 °C. Import was analyzed as in Fig. 1.



The results of Fig. 2establish the proper assay conditions for testing the role of mitochondrial receptors in the import of COXVa, since under these conditions the encounter of the precursor with the mitochondrial surface limits the rate of import. We therefore used these conditions to retest mitochondria pretreated with trypsin for import of various precursor proteins. Import of all precursors tested, including that of COXVa, was now strongly inhibited by trypsin pretreatment (Fig. 3).


Figure 3: Inhibition of COXVa import by trypsin pretreatment of mitochondria. The import rate was measured for the precursors to F (at 25 °C), COXVa (at 13 °C), HSP60 (at 10 °C), or for SU9-DHFR (at 25 °C); the concentration of mitochondria in the assay was 0.03 mg/ml, and samples were withdrawn at the indicated times. Where indicated, the mitochondria had been pretreated with 100 µg/ml trypsin. Import was analyzed as described in Fig. 1.



Inhibition of COXVa import by trypsin pretreatment of the mitochondria correlated with the degradation of the known import receptor subunits (Fig. 4A). At low concentrations, trypsin completely released Mas37p and Mas70p from the mitochondria, clipped the C terminus of Mas20p, and slightly reduced the import of COXVa. At higher trypsin concentrations, Mas20p and Mas22p were completely degraded and COXVa import was severely inhibited.


Figure 4: Import of COXVa is inhibited by degradation of import receptor subunits with trypsin or by antibodies against Mas20p. A, mitochondria were pretreated with the indicated amounts of trypsin. One aliquot of treated and untreated mitochondria was diluted to 0.03 mg/ml into import buffer and assayed for import of F (5 min at 25 °C; hatchedbars), COXVa (2 min at 13 °C; blackbars), or SU9-DHFR (2 min at 25 °C; openbars). Another aliquot of treated and untreated mitochondria (100 µg of mitochondrial protein) was analyzed by SDS-PAGE and immunoblotting for the protease-sensitive import receptor subunits Mas20p, Mas22p, Mas37p, and Mas70p, for the protease-resistant import site protein Isp42p, and for the intermembrane space marker cytochrome b. A sample of mitochondria (MP) that had been treated with 100 µg/ml trypsin in a hypoosmotic buffer to disrupt the outer membrane served as a control to show that the outer membrane barrier had remained intact under the conditions of trypsin treatment of mitochondria, protecting the highly trypsin-sensitive intermembrane space protein cytochrome b from the added protease (22). B, mitochondria (10 µg) were pretreated in a final volume of 0.1 ml with 100 µg of IgGs against porin, Mas20p, or Mas70p, diluted into import buffer to a final concentration of 0.03 mg/ml, and assayed for import of F (5 min at 25 °C, hatchedbars), COXVa (2 min at 13 °C, blackbars), or SU9-DHFR (2 min at 25 °C, openbars).



Import of COXVa was also inhibited by pretreatment of mitochondria with IgGs against the import receptor subunit Mas20p (Fig. 4B). IgGs against the Mas70p receptor subunit had no effect. As pointed out here and earlier(11) , a lack of inhibition in import assays cannot be reliably interpreted, and we do not conclude that import of COXVa bypasses the function of Mas70p. However, we do conclude that the Mas20p subunit is involved and that import of the COXVa precursor, like that of all other precursors that use the general import machinery, is initiated by the protein import receptor.


FOOTNOTES

*
This study was supported by grants from the Swiss National Science Foundation (31-40510.94) and from the Human Capital and Mobility Program of the European Economic Union (to G. S.) and by a fellowship from the Human Frontiers Science Program Organization (to T. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 41 61 267 2175.

The abbreviations used are: COXVa, subunit Va of yeast cytochrome c oxidase; SU9-DHFR, fusion protein between the presequence of subunit 9 of Neurospora crassa F-ATPase and dihydrofolate reductase; HSP60, heat shock protein 60; F, -subunit of the F-ATPase; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Mike Cumsky for the plasmid pDpT7-5a encoding COXVa, Sabine Rospert for the plasmid SP6-HSP60, Klaus Pfanner and Mike Cumsky for discussion of this work, Renate Looser and Hildegard Brütsch for technical assistance, Verena Grieder, Margrit Jäggi, and Liselotte Müller for photography, and members of the Schatz laboratory for critical reading of the manuscript.


REFERENCES
  1. Schatz, G.(1993) Protein Sci.2, 141-146 [Free Full Text]
  2. Kiebler, M., Becker, K., Pfanner, N. & Neupert, W.(1994) J. Membr. Biol.135, 191-207
  3. Söllner, T., Griffiths, G., Pfaller, R., Pfanner, N. & Neupert, W.(1989) Cell59, 1061-1070 [Medline] [Order article via Infotrieve]
  4. Steger, H. F., Söllner, T., Kiebler, M., Dietmeier, K. A., Pfaller, R., Trülzsch, K. S., Tropschug, M., Neupert, W. & Pfanner, N.(1990) J. Cell Biol.111, 2353-2363 [Abstract]
  5. Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brütsch, H. & Schatz, G.(1990) EMBO J.9, 3191-3200 [Abstract]
  6. Ramage, L., Junne, T., Hahne, K., Lithgow, T. & Schatz, G.(1993) EMBO J.12, 4115-4123 [Abstract]
  7. Kiebler, M., Keil, P., Schneider, H., van der Klei, I. J., Pfanner, N. & Neupert, W.(1993) Cell74, 483-492 [Medline] [Order article via Infotrieve]
  8. Moczko, M., Ehmann, B., Gärtner, F., Hönlinger, A., Schäfer, E. & Pfanner, N.(1994) J. Biol. Chem.269, 9045-9051 [Abstract/Free Full Text]
  9. Gratzer, S., Lithgow, T., Bauer, R. E., Lamping, E., Paltauf, F., Kohlwein, S. D., Haucke, V., Junne, T., Schatz, G. & Horst, M. (1995) J. Cell Biol.129, 25-34 [Abstract]
  10. Pfanner, N., Müller, H. K., Harmey, M. A. & Neupert, W.(1987) EMBO J.6, 3449-3454 [Abstract]
  11. Hines, V. & Schatz, G.(1993) J. Biol. Chem268, 449-454 [Abstract/Free Full Text]
  12. Haucke, V., Lithgow, T., Rospert, S., Hahne, K. & Schatz, G.(1995) J. Biol. Chem.270, 5565-5570 [Abstract/Free Full Text]
  13. Söllner, T., Rassow, J., Wiedman, J., Schlossman, J., Keil, P., Neupert, W. & Pfanner, N.(1992) Nature355, 84-87 [CrossRef][Medline] [Order article via Infotrieve]
  14. Lithgow, T., Junne, T., Suda, K., Gratzer, S. & Schatz, G.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 11973-11977 [Abstract/Free Full Text]
  15. Cumsky, M. G., Trueblood, C. E., Ko, C. & Poynton, R. O.(1987) Mol. Cell. Biol.7, 3511-3519 [Medline] [Order article via Infotrieve]
  16. Miller, B. R. & Cumsky, M. G.(1991) J. Cell Biol112, 833-841 [Abstract]
  17. Gärtner, F., Voos, W., Querol, A., Miller, B. R., Craig, E. A., Cumsky, M. G. & Pfanner, N.(1995) J. Biol. Chem.270, 3788-3795 [Abstract/Free Full Text]
  18. Glick, B. S. & Pon, L. A.(1995) Methods Enzymol., in press
  19. Schägger, H. & von Jagow, G.(1987) Anal. Biochem.166, 368-379 [Medline] [Order article via Infotrieve]
  20. Lill, R., Stuart, R. A., Drygas, M. E., Nargang, F. E. & Neupert, W. (1992) EMBO J.11, 449-456 [Abstract]
  21. Lithgow, T., Junne, T., Wachter, C. & Schatz, G.(1994) J. Biol. Chem.269, 15325-15330 [Abstract/Free Full Text]
  22. Glick, B. S., Brandt, A., Cunningham, K., Müller, S., Hallberg, R. L. & Schatz, G.(1992) Cell69, 809-822 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.