Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Dec 1;111(6):2341–2351. doi: 10.1083/jcb.111.6.2341

Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1

PMCID: PMC2116406  PMID: 1703535

Abstract

We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C.J., P.J. Cooper, P.J. Wise, and P.R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly----Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro----Leu and Ile----Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Arakawa H., Takiguchi M., Amaya Y., Nagata S., Hayashi H., Mori M. cDNA-derived amino acid sequence of rat mitochondrial 3-oxoacyl-CoA thiolase with no transient presequence: structural relationship with peroxisomal isozyme. EMBO J. 1987 May;6(5):1361–1366. doi: 10.1002/j.1460-2075.1987.tb02376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barlow D. J., Thornton J. M. Helix geometry in proteins. J Mol Biol. 1988 Jun 5;201(3):601–619. doi: 10.1016/0022-2836(88)90641-9. [DOI] [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Borst P. How proteins get into microbodies (peroxisomes, glyoxysomes, glycosomes). Biochim Biophys Acta. 1986 May 5;866(4):179–203. doi: 10.1016/0167-4781(86)90044-8. [DOI] [PubMed] [Google Scholar]
  6. Borst P. Peroxisome biogenesis revisited. Biochim Biophys Acta. 1989 Jun 1;1008(1):1–13. doi: 10.1016/0167-4781(89)90163-2. [DOI] [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Danpure C. J., Jennings P. R. Further studies on the activity and subcellular distribution of alanine:glyoxylate aminotransferase in the livers of patients with primary hyperoxaluria type 1. Clin Sci (Lond) 1988 Sep;75(3):315–322. doi: 10.1042/cs0750315. [DOI] [PubMed] [Google Scholar]
  12. Danpure C. J., Jennings P. R. Peroxisomal alanine:glyoxylate aminotransferase deficiency in primary hyperoxaluria type I. FEBS Lett. 1986 May 26;201(1):20–24. doi: 10.1016/0014-5793(86)80563-4. [DOI] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  14. Gould S. G., Keller G. A., Subramani S. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol. 1987 Dec;105(6 Pt 2):2923–2931. doi: 10.1083/jcb.105.6.2923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gould S. J., Keller G. A., Schneider M., Howell S. H., Garrard L. J., Goodman J. M., Distel B., Tabak H., Subramani S. Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J. 1990 Jan;9(1):85–90. doi: 10.1002/j.1460-2075.1990.tb08083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gould S. J., Keller G. A., Subramani S. Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol. 1988 Sep;107(3):897–905. doi: 10.1083/jcb.107.3.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Helms C., Graham M. Y., Dutchik J. E., Olson M. V. A new method for purifying lambda DNA from phage lysates. DNA. 1985 Feb;4(1):39–49. doi: 10.1089/dna.1985.4.39. [DOI] [PubMed] [Google Scholar]
  19. Lemire B. D., Fankhauser C., Baker A., Schatz G. The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity. J Biol Chem. 1989 Dec 5;264(34):20206–20215. [PubMed] [Google Scholar]
  20. Loenen W. A., Brammar W. J. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. doi: 10.1016/0378-1119(80)90054-2. [DOI] [PubMed] [Google Scholar]
  21. Miyazawa S., Osumi T., Hashimoto T., Ohno K., Miura S., Fujiki Y. Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mol Cell Biol. 1989 Jan;9(1):83–91. doi: 10.1128/mcb.9.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Noguchi T., Takada Y., Oota Y. Intraperoxisomal and intramitochondrial localization, and assay of pyruvate (glyoxylate) aminotransferase from rat liver. Hoppe Seylers Z Physiol Chem. 1979 Jul;360(7):919–927. doi: 10.1515/bchm2.1979.360.2.919. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Oda T., Miyajima H., Suzuki Y., Ichiyama A. Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat liver. Eur J Biochem. 1987 Nov 2;168(3):537–542. doi: 10.1111/j.1432-1033.1987.tb13451.x. [DOI] [PubMed] [Google Scholar]
  25. Okuno E., Minatogawa Y., Nakanishi J., Nakamura M., Kamoda N., Makino M., Kido R. The subcellular distribution of alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase in dog liver. Biochem J. 1979 Sep 15;182(3):877–879. doi: 10.1042/bj1820877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Opperdoes F. R. Glycosomes may provide clues to the import of peroxisomal proteins. Trends Biochem Sci. 1988 Jul;13(7):255–260. doi: 10.1016/0968-0004(88)90158-2. [DOI] [PubMed] [Google Scholar]
  27. Roise D., Schatz G. Mitochondrial presequences. J Biol Chem. 1988 Apr 5;263(10):4509–4511. [PubMed] [Google Scholar]
  28. Schatz G. 17th Sir Hans Krebs lecture. Signals guiding proteins to their correct locations in mitochondria. Eur J Biochem. 1987 May 15;165(1):1–6. doi: 10.1111/j.1432-1033.1987.tb11186.x. [DOI] [PubMed] [Google Scholar]
  29. Schiffer M., Edmundson A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967 Mar;7(2):121–135. doi: 10.1016/S0006-3495(67)86579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Small G. M., Szabo L. J., Lazarow P. B. Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes. EMBO J. 1988 Apr;7(4):1167–1173. doi: 10.1002/j.1460-2075.1988.tb02927.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Takada Y., Noguchi T. The evolution of peroxisomal and mitochondrial alanine: glyoxylate aminotransferase 1 in mammalian liver. Biochem Biophys Res Commun. 1982 Sep 16;108(1):153–157. doi: 10.1016/0006-291x(82)91844-7. [DOI] [PubMed] [Google Scholar]
  33. Wise P. J., Danpure C. J., Jennings P. R. Immunological heterogeneity of hepatic alanine:glyoxylate aminotransferase in primary hyperoxaluria type 1. FEBS Lett. 1987 Sep 28;222(1):17–20. doi: 10.1016/0014-5793(87)80183-7. [DOI] [PubMed] [Google Scholar]
  34. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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