Abstract
We have studied the post-translational import of incomplete precursor chains into isolated yeast mitochondria. The precursor was a fusion protein containing a mitochondrial presequence attached to mouse dihydrofolate reductase. In vitro-synthesis of the precursor was interrupted by the elongation inhibitor cycloheximide and the arrested nascent chains cosedimenting with ribosomes were released by EDTA. These incomplete chains were efficiently imported by isolated yeast mitochondria; their import resembled that of the complete precursor in requiring an energized inner membrane and a mitochondrial presequence. It differed from that of the completed precursor in its resistance to methotrexate (which only binds to correctly folded dihydrofolate reductase) and its independence of added ATP. The incomplete chains were also more sensitive to proteinase K than the completed precursor. We conclude that the incomplete chains were incompletely folded and suggest that the lack of tight folding caused import into mitochondria to become independent of added ATP. This implies that ATP may participate, directly or indirectly, in the unfolding of the precursor for its transport into mitochondria.
Full text
PDF







Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ades I. Z., Butow R. A. The products of mitochondria-bound cytoplasmic polysomes in yeast. J Biol Chem. 1980 Oct 25;255(20):9918–9924. [PubMed] [Google Scholar]
- Chen L., Tai P. C. ATP is essential for protein translocation into Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4384–4388. doi: 10.1073/pnas.82.13.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chien S. M., Freeman K. B. Mitochondrial malate dehydrogenase and its precursor have different conformations. Biochem Biophys Res Commun. 1986 Nov 26;141(1):313–318. doi: 10.1016/s0006-291x(86)80370-9. [DOI] [PubMed] [Google Scholar]
- Colombo B., Felicetti L., Baglioni C. Inhibition of protein synthesis in reticulocytes by antibiotics. I. Effects on polysomes. Biochim Biophys Acta. 1966 Apr 18;119(1):109–119. doi: 10.1016/0005-2787(66)90043-8. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
- Flügge U. I., Hinz G. Energy dependence of protein translocation into chloroplasts. Eur J Biochem. 1986 Nov 3;160(3):563–570. doi: 10.1111/j.1432-1033.1986.tb10075.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Geller B. L., Movva N. R., Wickner W. Both ATP and the electrochemical potential are required for optimal assembly of pro-OmpA into Escherichia coli inner membrane vesicles. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4219–4222. doi: 10.1073/pnas.83.12.4219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Godchaux W., 3rd, Adamson S. D., Herbert E. Effects of cycloheximide on polyribosome function in reticulocytes. J Mol Biol. 1967 Jul 14;27(1):57–72. doi: 10.1016/0022-2836(67)90351-8. [DOI] [PubMed] [Google Scholar]
- Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
- 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]
- Howell E. E., Villafranca J. E., Warren M. S., Oatley S. J., Kraut J. Functional role of aspartic acid-27 in dihydrofolate reductase revealed by mutagenesis. Science. 1986 Mar 7;231(4742):1123–1128. doi: 10.1126/science.3511529. [DOI] [PubMed] [Google Scholar]
- 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]
- Hurt E. C., Pesold-Hurt B., Schatz G. The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. FEBS Lett. 1984 Dec 10;178(2):306–310. doi: 10.1016/0014-5793(84)80622-5. [DOI] [PubMed] [Google Scholar]
- Kellems R. E., Allison V. F., Butow R. A. Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria. J Cell Biol. 1975 Apr;65(1):1–14. doi: 10.1083/jcb.65.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mueckler M., Lodish H. F. Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage. Nature. 1986 Aug 7;322(6079):549–552. doi: 10.1038/322549a0. [DOI] [PubMed] [Google Scholar]
- Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Rothblatt J. A., Meyer D. I. Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986 Feb 28;44(4):619–628. doi: 10.1016/0092-8674(86)90271-0. [DOI] [PubMed] [Google Scholar]
- Rothman J. E., Kornberg R. D. Cell biology. An unfolding story of protein translocation. Nature. 1986 Jul 17;322(6076):209–210. doi: 10.1038/322209a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Suissa M., Schatz G. Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes. J Biol Chem. 1982 Nov 10;257(21):13048–13055. [PubMed] [Google Scholar]
- Taniuchi H. Formation of randomly paired disulfide bonds in des-(121-124)-ribonuclease after reduction and reoxidation. J Biol Chem. 1970 Oct 25;245(20):5459–5468. [PubMed] [Google Scholar]
- Volz K. W., Matthews D. A., Alden R. A., Freer S. T., Hansch C., Kaufman B. T., Kraut J. Crystal structure of avian dihydrofolate reductase containing phenyltriazine and NADPH. J Biol Chem. 1982 Mar 10;257(5):2528–2536. [PubMed] [Google Scholar]
- Waters M. G., Blobel G. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol. 1986 May;102(5):1543–1550. doi: 10.1083/jcb.102.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]
- Wiedmann M., Huth A., Rapoport T. A. Xenopus oocytes can secrete bacterial beta-lactamase. Nature. 1984 Jun 14;309(5969):637–639. doi: 10.1038/309637a0. [DOI] [PubMed] [Google Scholar]
- 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]