Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Nov;7(11):4038–4047. doi: 10.1128/mcb.7.11.4038

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information.

D M Bedwell 1, D J Klionsky 1, S D Emr 1
PMCID: PMC368074  PMID: 2893254

Abstract

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.

Full text

PDF
4039

Images in this article

4041Image
on p.4041
4042Image
on p.4042
4042Image
on p.4042
4043Image
on p.4043
4043Image
on p.4043

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allison D. S., Schatz G. Artificial mitochondrial presequences. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9011–9015. doi: 10.1073/pnas.83.23.9011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  4. Chen W. J., Douglas M. G. Phosphodiester bond cleavage outside mitochondria is required for the completion of protein import into the mitochondrial matrix. Cell. 1987 Jun 5;49(5):651–658. doi: 10.1016/0092-8674(87)90541-1. [DOI] [PubMed] [Google Scholar]
  5. Daum G., Böhni P. C., Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13028–13033. [PubMed] [Google Scholar]
  6. Douglas M. G., Geller B. L., Emr S. D. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983–3987. doi: 10.1073/pnas.81.13.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Douglas M. G., McCammon M. T., Vassarotti A. Targeting proteins into mitochondria. Microbiol Rev. 1986 Jun;50(2):166–178. doi: 10.1128/mr.50.2.166-178.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eilers M., Oppliger W., Schatz G. Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria. EMBO J. 1987 Apr;6(4):1073–1077. doi: 10.1002/j.1460-2075.1987.tb04860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Emr S. D., Vassarotti A., Garrett J., Geller B. L., Takeda M., Douglas M. G. The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal. J Cell Biol. 1986 Feb;102(2):523–533. doi: 10.1083/jcb.102.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gasser S. M., Daum G., Schatz G. Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria. J Biol Chem. 1982 Nov 10;257(21):13034–13041. [PubMed] [Google Scholar]
  11. Gillespie L. L., Argan C., Taneja A. T., Hodges R. S., Freeman K. B., Shore G. C. A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix. J Biol Chem. 1985 Dec 25;260(30):16045–16048. [PubMed] [Google Scholar]
  12. Goldring E. S., Grossman L. I., Krupnick D., Cryer D. R., Marmur J. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol. 1970 Sep 14;52(2):323–335. doi: 10.1016/0022-2836(70)90033-1. [DOI] [PubMed] [Google Scholar]
  13. HALVORSON H., ELLIAS L. The purification and properties of an alpha-glucosidase of Saccharomyces italicus Y1225. Biochim Biophys Acta. 1958 Oct;30(1):28–40. doi: 10.1016/0006-3002(58)90237-3. [DOI] [PubMed] [Google Scholar]
  14. Hase T., Müller U., Riezman H., Schatz G. A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus. EMBO J. 1984 Dec 20;3(13):3157–3164. doi: 10.1002/j.1460-2075.1984.tb02274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hay R., Böhni P., Gasser S. How mitochondria import proteins. Biochim Biophys Acta. 1984 Jan 27;779(1):65–87. doi: 10.1016/0304-4157(84)90004-2. [DOI] [PubMed] [Google Scholar]
  16. Horwich A. L., Kalousek F., Fenton W. A., Pollock R. A., Rosenberg L. E. Targeting of pre-ornithine transcarbamylase to mitochondria: definition of critical regions and residues in the leader peptide. Cell. 1986 Feb 14;44(3):451–459. doi: 10.1016/0092-8674(86)90466-6. [DOI] [PubMed] [Google Scholar]
  17. Hurt E. C., Allison D. S., Müller U., Schatz G. Amino-terminal deletions in the presequence of an imported mitochondrial protein block the targeting function and proteolytic cleavage of the presequence at the carboxy terminus. J Biol Chem. 1987 Jan 25;262(3):1420–1424. [PubMed] [Google Scholar]
  18. Hurt E. C., Pesold-Hurt B., Suda K., Oppliger W., Schatz G. The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. EMBO J. 1985 Aug;4(8):2061–2068. doi: 10.1002/j.1460-2075.1985.tb03892.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnson L. M., Bankaitis V. A., Emr S. D. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. doi: 10.1016/0092-8674(87)90084-5. [DOI] [PubMed] [Google Scholar]
  21. Keng T., Alani E., Guarente L. The nine amino-terminal residues of delta-aminolevulinate synthase direct beta-galactosidase into the mitochondrial matrix. Mol Cell Biol. 1986 Feb;6(2):355–364. doi: 10.1128/mcb.6.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laris P. C. Evidence for the electrogenic nature of the ATP-ADP exchange system in rat liver mitochondria. Biochim Biophys Acta. 1977 Jan 6;459(1):110–118. doi: 10.1016/0005-2728(77)90013-5. [DOI] [PubMed] [Google Scholar]
  23. Maccecchini M. L., Rudin Y., Blobel G., Schatz G. Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc Natl Acad Sci U S A. 1979 Jan;76(1):343–347. doi: 10.1073/pnas.76.1.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  25. Newman A. J., Lin R. J., Cheng S. C., Abelson J. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell. 1985 Aug;42(1):335–344. doi: 10.1016/s0092-8674(85)80129-x. [DOI] [PubMed] [Google Scholar]
  26. Ohta S., Schatz G. A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria. EMBO J. 1984 Mar;3(3):651–657. doi: 10.1002/j.1460-2075.1984.tb01862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. PULLMAN M. E., PENEFSKY H. S., DATTA A., RACKER E. Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphatase. J Biol Chem. 1960 Nov;235:3322–3329. [PubMed] [Google Scholar]
  28. Pfaff E., Klingenberg M. Adenine nucleotide translocation of mitochondria. 1. Specificity and control. Eur J Biochem. 1968 Oct 17;6(1):66–79. doi: 10.1111/j.1432-1033.1968.tb00420.x. [DOI] [PubMed] [Google Scholar]
  29. Pfanner N., Neupert W. Transport of F1-ATPase subunit beta into mitochondria depends on both a membrane potential and nucleoside triphosphates. FEBS Lett. 1986 Dec 15;209(2):152–156. doi: 10.1016/0014-5793(86)81101-2. [DOI] [PubMed] [Google Scholar]
  30. Pfanner N., Neupert W. Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. EMBO J. 1985 Nov;4(11):2819–2825. doi: 10.1002/j.1460-2075.1985.tb04009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pilgrim D., Young E. T. Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space. Mol Cell Biol. 1987 Jan;7(1):294–304. doi: 10.1128/mcb.7.1.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reid G. A., Schatz G. Import of proteins into mitochondria. Extramitochondrial pools and post-translational import of mitochondrial protein precursors in vivo. J Biol Chem. 1982 Nov 10;257(21):13062–13067. [PubMed] [Google Scholar]
  33. Richardson W. D., Roberts B. L., Smith A. E. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. doi: 10.1016/0092-8674(86)90486-1. [DOI] [PubMed] [Google Scholar]
  34. Riezman H., Hay R., Witte C., Nelson N., Schatz G. Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins. EMBO J. 1983;2(7):1113–1118. doi: 10.1002/j.1460-2075.1983.tb01554.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roise D., Horvath S. J., Tomich J. M., Richards J. H., Schatz G. A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. EMBO J. 1986 Jun;5(6):1327–1334. doi: 10.1002/j.1460-2075.1986.tb04363.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Saltzgaber-Muller J., Kunapuli S. P., Douglas M. G. Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit. J Biol Chem. 1983 Oct 10;258(19):11465–11470. [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schleyer M., Neupert W. Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell. 1985 Nov;43(1):339–350. doi: 10.1016/0092-8674(85)90039-x. [DOI] [PubMed] [Google Scholar]
  39. Szekely E., Montgomery D. L. Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components. Mol Cell Biol. 1984 May;4(5):939–946. doi: 10.1128/mcb.4.5.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takeda M., Vassarotti A., Douglas M. G. Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor. J Biol Chem. 1985 Dec 15;260(29):15458–15465. [PubMed] [Google Scholar]
  41. Vassarotti A., Chen W. J., Smagula C., Douglas M. G. Sequences distal to the mitochondrial targeting sequences are necessary for the maturation of the F1-ATPase beta-subunit precursor in mitochondria. J Biol Chem. 1987 Jan 5;262(1):411–418. [PubMed] [Google Scholar]
  42. Vassarotti A., Stroud R., Douglas M. Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import. EMBO J. 1987 Mar;6(3):705–711. doi: 10.1002/j.1460-2075.1987.tb04811.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wickerham L. J. A Critical Evaluation of the Nitrogen Assimilation Tests Commonly Used in the Classification of Yeasts. J Bacteriol. 1946 Sep;52(3):293–301. [PMC free article] [PubMed] [Google Scholar]
  44. Zimmerman R., Paluch U., Sprinzl M., Neupert W. Cell-free synthesis of the mitochondrial ADP/ATP carrier protein of Neurospora crassa. Eur J Biochem. 1979 Sep;99(2):247–252. doi: 10.1111/j.1432-1033.1979.tb13251.x. [DOI] [PubMed] [Google Scholar]
  45. Zwizinski C., Schleyer M., Neupert W. Proteinaceous receptors for the import of mitochondrial precursor proteins. J Biol Chem. 1984 Jun 25;259(12):7850–7856. [PubMed] [Google Scholar]
  46. van Loon A. P., Brändli A. W., Schatz G. The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting. Cell. 1986 Mar 14;44(5):801–812. doi: 10.1016/0092-8674(86)90846-9. [DOI] [PubMed] [Google Scholar]
  47. von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 1986 Jun;5(6):1335–1342. doi: 10.1002/j.1460-2075.1986.tb04364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES