Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Nov 2;135(4):939–951. doi: 10.1083/jcb.135.4.939

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

PMCID: PMC2133393  PMID: 8922378

Abstract

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.

Full Text

The Full Text of this article is available as a PDF (4.1 MB).

Selected References

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

  1. Attardi G., Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol. 1988;4:289–333. doi: 10.1146/annurev.cb.04.110188.001445. [DOI] [PubMed] [Google Scholar]
  2. Brul S., Wiemer E. A., Westerveld A., Strijland A., Wanders R. J., Schram A. W., Heymans H. S., Schutgens R. B., Van den Bosch H., Tager J. M. Kinetics of the assembly of peroxisomes after fusion of complementary cell lines from patients with the cerebro-hepato-renal (Zellweger) syndrome and related disorders. Biochem Biophys Res Commun. 1988 May 16;152(3):1083–1089. doi: 10.1016/s0006-291x(88)80395-4. [DOI] [PubMed] [Google Scholar]
  3. Chen W. J., Douglas M. G. An F1-ATPase beta-subunit precursor lacking an internal tetramer-forming domain is imported into mitochondria in the absence of ATP. J Biol Chem. 1988 Apr 15;263(11):4997–5000. [PubMed] [Google Scholar]
  4. Chen W. J., Douglas M. G. The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation. J Biol Chem. 1987 Nov 15;262(32):15605–15609. [PubMed] [Google Scholar]
  5. Cooper P. J., Danpure C. J., Wise P. J., Guttridge K. M. Immunocytochemical localization of human hepatic alanine: glyoxylate aminotransferase in control subjects and patients with primary hyperoxaluria type 1. J Histochem Cytochem. 1988 Oct;36(10):1285–1294. doi: 10.1177/36.10.3418107. [DOI] [PubMed] [Google Scholar]
  6. Danpure C. J., Cooper P. J., Wise P. J., Jennings P. R. An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria. J Cell Biol. 1989 Apr;108(4):1345–1352. doi: 10.1083/jcb.108.4.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Danpure C. J., Fryer P., Griffiths S., Guttridge K. M., Jennings P. R., Allsop J., Moser A. B., Naidu S., Moser H. W., MacCollin M. Cytosolic compartmentalization of hepatic alanine:glyoxylate aminotransferase in patients with aberrant peroxisomal biogenesis and its effect on oxalate metabolism. J Inherit Metab Dis. 1994;17(1):27–40. doi: 10.1007/BF00735393. [DOI] [PubMed] [Google Scholar]
  8. Eilers M., Schatz G. Protein unfolding and the energetics of protein translocation across biological membranes. Cell. 1988 Feb 26;52(4):481–483. doi: 10.1016/0092-8674(88)90458-8. [DOI] [PubMed] [Google Scholar]
  9. Frydman J., Nimmesgern E., Ohtsuka K., Hartl F. U. Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature. 1994 Jul 14;370(6485):111–117. doi: 10.1038/370111a0. [DOI] [PubMed] [Google Scholar]
  10. Glover J. R., Andrews D. W., Rachubinski R. A. Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10541–10545. doi: 10.1073/pnas.91.22.10541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goodman J. M., Scott C. W., Donahue P. N., Atherton J. P. Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii. J Biol Chem. 1984 Jul 10;259(13):8485–8493. [PubMed] [Google Scholar]
  12. Gould S. J., Keller G. A., Hosken N., Wilkinson J., Subramani S. A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol. 1989 May;108(5):1657–1664. doi: 10.1083/jcb.108.5.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hartl F. U., Pfanner N., Nicholson D. W., Neupert W. Mitochondrial protein import. Biochim Biophys Acta. 1989 Jan 18;988(1):1–45. doi: 10.1016/0304-4157(89)90002-6. [DOI] [PubMed] [Google Scholar]
  14. Hendrick J. P., Langer T., Davis T. A., Hartl F. U., Wiedmann M. Control of folding and membrane translocation by binding of the chaperone DnaJ to nascent polypeptides. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10216–10220. doi: 10.1073/pnas.90.21.10216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lazarow P. B., Fujiki Y. Biogenesis of peroxisomes. Annu Rev Cell Biol. 1985;1:489–530. doi: 10.1146/annurev.cb.01.110185.002421. [DOI] [PubMed] [Google Scholar]
  16. Lazarow P. B., de Duve C. The synthesis and turnover of rat liver of rat liver peroxisomes. IV. Biochemical pathway of catalase synthesis. J Cell Biol. 1973 Nov;59(2 Pt 1):491–506. doi: 10.1083/jcb.59.2.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lazarow P. B., de Duve C. The synthesis and turnover of rat liver peroxisomes. V. Intracellular pathway of catalase synthesis. J Cell Biol. 1973 Nov;59(2 Pt 1):507–524. doi: 10.1083/jcb.59.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McNew J. A., Goodman J. M. An oligomeric protein is imported into peroxisomes in vivo. J Cell Biol. 1994 Dec;127(5):1245–1257. doi: 10.1083/jcb.127.5.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Middelkoop E., Strijland A., Tager J. M. Does aminotriazole inhibit import of catalase into peroxisomes by retarding unfolding? FEBS Lett. 1991 Feb 11;279(1):79–82. doi: 10.1016/0014-5793(91)80255-2. [DOI] [PubMed] [Google Scholar]
  20. Middelkoop E., Wiemer E. A., Schoenmaker D. E., Strijland A., Tager J. M. Topology of catalase assembly in human skin fibroblasts. Biochim Biophys Acta. 1993 Dec 16;1220(1):15–20. doi: 10.1016/0167-4889(93)90091-3. [DOI] [PubMed] [Google Scholar]
  21. Motley A., Lumb M. J., Oatey P. B., Jennings P. R., De Zoysa P. A., Wanders R. J., Tabak H. F., Danpure C. J. Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1. J Cell Biol. 1995 Oct;131(1):95–109. doi: 10.1083/jcb.131.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Noguchi T., Takada Y. Peroxisomal localization of alanine: glyoxylate aminotransferase in human liver. Arch Biochem Biophys. 1979 Sep;196(2):645–647. doi: 10.1016/0003-9861(79)90319-9. [DOI] [PubMed] [Google Scholar]
  23. Nye S. H., Scarpulla R. C. Mitochondrial targeting of yeast apoiso-1-cytochrome c is mediated through functionally independent structural domains. Mol Cell Biol. 1990 Nov;10(11):5763–5771. doi: 10.1128/mcb.10.11.5763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Osumi T., Tsukamoto T., Hata S., Yokota S., Miura S., Fujiki Y., Hijikata M., Miyazawa S., Hashimoto T. Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem Biophys Res Commun. 1991 Dec 31;181(3):947–954. doi: 10.1016/0006-291x(91)92028-i. [DOI] [PubMed] [Google Scholar]
  25. Pfanner N., Neupert W. The mitochondrial protein import apparatus. Annu Rev Biochem. 1990;59:331–353. doi: 10.1146/annurev.bi.59.070190.001555. [DOI] [PubMed] [Google Scholar]
  26. Purdue P. E., Allsop J., Isaya G., Rosenberg L. E., Danpure C. J. Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10900–10904. doi: 10.1073/pnas.88.23.10900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Purdue P. E., Takada Y., Danpure C. J. Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1. J Cell Biol. 1990 Dec;111(6 Pt 1):2341–2351. doi: 10.1083/jcb.111.6.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roise D., Schatz G. Mitochondrial presequences. J Biol Chem. 1988 Apr 5;263(10):4509–4511. [PubMed] [Google Scholar]
  29. 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]
  30. Subramani S. Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol. 1993;9:445–478. doi: 10.1146/annurev.cb.09.110193.002305. [DOI] [PubMed] [Google Scholar]
  31. Swinkels B. W., Gould S. J., Bodnar A. G., Rachubinski R. A., Subramani S. A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J. 1991 Nov;10(11):3255–3262. doi: 10.1002/j.1460-2075.1991.tb04889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Swinkels B. W., Gould S. J., Subramani S. Targeting efficiencies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett. 1992 Jun 29;305(2):133–136. doi: 10.1016/0014-5793(92)80880-p. [DOI] [PubMed] [Google Scholar]
  33. Takada Y., Kaneko N., Esumi H., Purdue P. E., Danpure C. J. Human peroxisomal L-alanine: glyoxylate aminotransferase. Evolutionary loss of a mitochondrial targeting signal by point mutation of the initiation codon. Biochem J. 1990 Jun 1;268(2):517–520. doi: 10.1042/bj2680517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Takada Y., Noguchi T. Subcellular distribution, and physical and immunological properties of hepatic alanine: glyoxylate aminotransferase isoenzymes in different mammalian species. Comp Biochem Physiol B. 1982;72(4):597–604. doi: 10.1016/0305-0491(82)90512-0. [DOI] [PubMed] [Google Scholar]
  35. Thompson J. S., Richardson K. E. Isolation and characterization of an L-alanine: glyoxylate aminotransferase from human liver. J Biol Chem. 1967 Aug 25;242(16):3614–3619. [PubMed] [Google Scholar]
  36. Walton P. A., Hill P. E., Subramani S. Import of stably folded proteins into peroxisomes. Mol Biol Cell. 1995 Jun;6(6):675–683. doi: 10.1091/mbc.6.6.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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 Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES