Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1987 Oct;6(10):3117–3123. doi: 10.1002/j.1460-2075.1987.tb02621.x

The interaction of a synthetic mitochondrial signal peptide with lipid membranes is independent of transbilayer potential.

I S Skerjanc 1, G C Shore 1, J R Silvius 1
PMCID: PMC553752  PMID: 3691482

Abstract

We have used fluorescence measurements and assays of vesicle disruption (contents leakage) to monitor the interaction between lipid vesicles and a synthetic peptide corresponding to the N-terminal 27 amino acids of rat mitochondrial pre-ornithine carbamyltransferase (pOCT). This peptide and two fluorescent derivatives bind reversibly to vesicles composed of neutral and anionic phospholipids with increasing affinity as the proportion of anionic lipids in the vesicles increases. The affinity of the peptide for lipid vesicles is unaffected by the presence of a transbilayer potential (inside negative) of at least -80 mV across the vesicle membranes. Our results support the proposal that the signal sequence of pOCT may promote an initial association of the precursor protein with mitochondrial membranes prior to binding to a specific receptor. However, we find no evidence that the pOCT signal sequence can subsequently undergo transfer into or across the lipid bilayer, even in the presence of a transmembrane potential of the magnitude previously found to support the import of precursor proteins into mitochondria.

Full text

PDF
3117

Selected References

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

  1. Allen T. M., Cleland L. G. Serum-induced leakage of liposome contents. Biochim Biophys Acta. 1980 Apr 10;597(2):418–426. doi: 10.1016/0005-2736(80)90118-2. [DOI] [PubMed] [Google Scholar]
  2. Bernheimer A. W., Rudy B. Interactions between membranes and cytolytic peptides. Biochim Biophys Acta. 1986 Jun 12;864(1):123–141. doi: 10.1016/0304-4157(86)90018-3. [DOI] [PubMed] [Google Scholar]
  3. Comfurius P., Zwaal R. F. The enzymatic synthesis of phosphatidylserine and purification by CM-cellulose column chromatography. Biochim Biophys Acta. 1977 Jul 20;488(1):36–42. doi: 10.1016/0005-2760(77)90120-5. [DOI] [PubMed] [Google Scholar]
  4. Daum G. Lipids of mitochondria. Biochim Biophys Acta. 1985 Jun 12;822(1):1–42. doi: 10.1016/0304-4157(85)90002-4. [DOI] [PubMed] [Google Scholar]
  5. DeGrado W. F., Musso G. F., Lieber M., Kaiser E. T., Kézdy F. J. Kinetics and mechanism of hemolysis induced by melittin and by a synthetic melittin analogue. Biophys J. 1982 Jan;37(1):329–338. doi: 10.1016/S0006-3495(82)84681-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Epand R. M., Hui S. W., Argan C., Gillespie L. L., Shore G. C. Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide. J Biol Chem. 1986 Aug 5;261(22):10017–10020. [PubMed] [Google Scholar]
  8. Eytan G. D., Almary T. Melittin-induced fusion of acidic liposomes. FEBS Lett. 1983 May 30;156(1):29–32. doi: 10.1016/0014-5793(83)80241-5. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hanspal M., Ralston G. B. Purification of a trypsin-insensitive fragment of spectrin from human erythrocyte membranes. Biochim Biophys Acta. 1981 Jul 28;669(2):133–139. doi: 10.1016/0005-2795(81)90234-8. [DOI] [PubMed] [Google Scholar]
  11. Hennig B., Koehler H., Neupert W. Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4963–4967. doi: 10.1073/pnas.80.16.4963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hermetter A., Lakowicz J. R. The aggregation state of mellitin in lipid bilayers. An energy transfer study. J Biol Chem. 1986 Jun 25;261(18):8243–8248. [PubMed] [Google Scholar]
  13. Horwich A. L., Kalousek F., Mellman I., Rosenberg L. E. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J. 1985 May;4(5):1129–1135. doi: 10.1002/j.1460-2075.1985.tb03750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hurt E. C., Müller U., Schatz G. The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane. EMBO J. 1985 Dec 16;4(13A):3509–3518. doi: 10.1002/j.1460-2075.1985.tb04110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hurt E. C., Pesold-Hurt B., Schatz G. The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984 Dec 20;3(13):3149–3156. doi: 10.1002/j.1460-2075.1984.tb02272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ito A., Ogishima T., Ou W., Omura T., Aoyagi H., Lee S., Mihara H., Izumiya N. Effects of synthetic model peptides resembling the extension peptides of mitochondrial enzyme precursors on import of the precursors into mitochondria. J Biochem. 1985 Dec;98(6):1571–1582. doi: 10.1093/oxfordjournals.jbchem.a135426. [DOI] [PubMed] [Google Scholar]
  17. Kanellis P., Romans A. Y., Johnson B. J., Kercret H., Chiovetti R., Jr, Allen T. M., Segrest J. P. Studies of synthetic peptide analogs of the amphipathic helix. Effect of charged amino acid residue topography on lipid affinity. J Biol Chem. 1980 Dec 10;255(23):11464–11472. [PubMed] [Google Scholar]
  18. Kempf C., Klausner R. D., Weinstein J. N., Van Renswoude J., Pincus M., Blumenthal R. Voltage-dependent trans-bilayer orientation of melittin. J Biol Chem. 1982 Mar 10;257(5):2469–2476. [PubMed] [Google Scholar]
  19. 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]
  20. Lingelbach K. R., Graf L. J., Dunn A. R., Hoogenraad N. J. Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import. Eur J Biochem. 1986 Nov 17;161(1):19–23. doi: 10.1111/j.1432-1033.1986.tb10119.x. [DOI] [PubMed] [Google Scholar]
  21. McIntyre P., Graf L., Mercer J., Peterson G., Hudson P., Hoogenraad N. A highly basic N-terminal extension of the mitochondrial matrix enzyme ornithine transcarbamylase from rat liver. FEBS Lett. 1984 Nov 5;177(1):41–46. doi: 10.1016/0014-5793(84)80977-1. [DOI] [PubMed] [Google Scholar]
  22. Morgan C. G., Williamson H., Fuller S., Hudson B. Melittin induces fusion of unilamellar phospholipid vesicles. Biochim Biophys Acta. 1983 Aug 10;732(3):668–674. doi: 10.1016/0005-2736(83)90245-6. [DOI] [PubMed] [Google Scholar]
  23. Nguyen M., Argan C., Lusty C. J., Shore G. C. Import and processing of hybrid proteins by mammalian mitochondria in vitro. J Biol Chem. 1986 Jan 15;261(2):800–805. [PubMed] [Google Scholar]
  24. Nguyen M., Argan C., Sheffield W. P., Bell A. W., Shields D., Shore G. C. A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase. J Cell Biol. 1987 May;104(5):1193–1198. doi: 10.1083/jcb.104.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Op den Kamp J. A. Lipid asymmetry in membranes. Annu Rev Biochem. 1979;48:47–71. doi: 10.1146/annurev.bi.48.070179.000403. [DOI] [PubMed] [Google Scholar]
  26. Papahadjopoulos D., Miller N. Phospholipid model membranes. I. Structural characteristics of hydrated liquid crystals. Biochim Biophys Acta. 1967 Sep 9;135(4):624–638. doi: 10.1016/0005-2736(67)90094-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Pick U. Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Arch Biochem Biophys. 1981 Nov;212(1):186–194. doi: 10.1016/0003-9861(81)90358-1. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Sandeaux R., Seta P., Jeminet G., Alleaume M., Gavach C. The influence of pH on the conductance of lipid bimolecular membranes in relation to the alkaline ion transport induced by carboxylic carriers grisorixin, alborixin and monensin. Biochim Biophys Acta. 1978 Aug 17;511(3):499–508. doi: 10.1016/0005-2736(78)90284-5. [DOI] [PubMed] [Google Scholar]
  32. Sargent D. F., Schwyzer R. Membrane lipid phase as catalyst for peptide-receptor interactions. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5774–5778. doi: 10.1073/pnas.83.16.5774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schwerzmann K., Cruz-Orive L. M., Eggman R., Sänger A., Weibel E. R. Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study. J Cell Biol. 1986 Jan;102(1):97–103. doi: 10.1083/jcb.102.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sims P. J., Waggoner A. S., Wang C. H., Hoffman J. F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315–3330. doi: 10.1021/bi00713a022. [DOI] [PubMed] [Google Scholar]
  35. Struck D. K., Hoekstra D., Pagano R. E. Use of resonance energy transfer to monitor membrane fusion. Biochemistry. 1981 Jul 7;20(14):4093–4099. doi: 10.1021/bi00517a023. [DOI] [PubMed] [Google Scholar]
  36. Tamm L. K. Incorporation of a synthetic mitochondrial signal peptide into charged and uncharged phospholipid monolayers. Biochemistry. 1986 Nov 18;25(23):7470–7476. doi: 10.1021/bi00371a032. [DOI] [PubMed] [Google Scholar]
  37. Vogel H., Jähnig F. The structure of melittin in membranes. Biophys J. 1986 Oct;50(4):573–582. doi: 10.1016/S0006-3495(86)83497-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yianni Y. P., Fitton J. E., Morgan C. G. Lytic effects of melittin and delta-haemolysin from Staphylococcus aureus on vesicles of dipalmitoylphosphatidylcholine. Biochim Biophys Acta. 1986 Mar 27;856(1):91–100. doi: 10.1016/0005-2736(86)90014-3. [DOI] [PubMed] [Google Scholar]
  39. Zwizinski C., Schleyer M., Neupert W. Transfer of proteins into mitochondria. Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria. J Biol Chem. 1983 Apr 10;258(7):4071–4074. [PubMed] [Google Scholar]
  40. 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]
  41. 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 The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES