Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1.
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.
Leiper
J M
JM
Medical Research Council Laboratory for Molecular Cell Biology, University College London, United Kingdom.
Oatey
P B
PB
Danpure
C J
CJ
D000410:Alanine Transaminase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000235:genetics; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells / Q000201:enzymology; D019281:Dimerization; D005998:Glycine / Q000235:genetics; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000201:enzymology*; D007930:Leucine / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D017354:Point Mutation / Q000502:physiology; D011110:Polymorphism, Genetic; D011392:Proline / Q000235:genetics; D000637:Transaminases
D001120:Arginine; D011392:Proline; D000637:Transaminases; D000410:Alanine Transaminase; C019635:Alanine-glyoxylate transaminase; D007930:Leucine; D005998:Glycine