INHIBITION OF ALANINE-GLYOXYLATE AMINOTRANSFERASE-1 DIMERIZATION IS APREREQUISITE FOR ITS PEROXISOME-TO-MITOCHONDRION MISTARGETING IN PRIMARY HYPEROXALURIA TYPE-1

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme ala nine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive di sease primary hyperoxaluria type 1 (PH1) is associated with the combin ed presence of a normally occurring Pro(11)Leu polymorphism and a PH1- specific Gly(170)Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which altho ugh sufficient to direct the import of in vitro-translated AGT into is olated mitochondria, requires the additional presence of the Gly(170)A rg mutation to function efficiently in whole cells. The role of this m utation in the mistargeting phenomenon has remained elusive. It does n ot interfere with the peroxisomal targeting or import of AGT. In the p resent study, we have investigated the role of the Gly(170)Arg mutatio n in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxis omal and mitochondrial import processes. The results obtained show tha t in vitro-translated AGT rapidly forms dimers that do not readily exc hange subunits. Although the presence of the Pro(11)Leu or Gly(170)Arg substitutions alone had no effect on dimerization, their combined pre sence abolished homodimerization in vitro. However, AGT containing bot h substitutions was still able to form heterodimers in vitro with eith er normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagge d and untagged forms of AGT in whole cells showed that normal AGT rapi dly dimerizes in the cytosol and is imported into peroxisomes as a dim er. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additi onal presence of the Gly(170)Arg substitution impairs dimerization suf ficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be i mported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.