In approximately one-third of primary hyperoxaluria type 1 patients, disease is associated with a unique protein sorting defect in which hepatic L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which is normally peroxisomal, is mistargeted to mitochondria. In all such patients analyzed to date, the gene encoding the aberrantly targeted AGT carries three point mutations, each of which specifies an amino acid substitution. In this paper we show that one of these substitutions, a proline-to-leucine at residue 11, is necessary and sufficient for the generation of a mitochondrial targeting sequence in the AGT protein. AGT with this substitution appears to interact specifically with the mitochondrial protein import machinery, via a discrete N-terminal domain of the AGT protein. The N-terminal 19 amino acids of AGT with this substitution are sufficient to direct mouse cytosolic dihydrofolate reductase to mitochondria, and a synthetic peptide corresponding to this same 19-amino acid region reversibly inhibits mitochondrial protein import, not only of AGT but also of ornithine transcarbamoylase, a genuine cytoplasmically synthesized mitochondrial protein. We have extended these studies to analyze a region of normal human AGT cDNA directly upstream of the coding region. This sequence appears to correspond to an ancestral mitochondrial targeting sequence deleted from the human coding region by point mutation at the initiation codon. We show that reestablishment ofthis initiation codon produces an active mitochondrial targeting sequence that is different to that found in the hyperoxaluria patients. These results are discussed with reference to the AGT targeting defect in primary hyperoxaluria and also in relation to the highly unusual species specificity of subcellular distribution of AGT among mammals.Primary hyperoxaluria type 1 (PHi) is a lethal autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44) (1). Whereas the majority ofPHi patients have a complete deficiency of AGT catalytic activity (2) and AGT immunoreactive protein (3), approximately one-third possess significant levels of residual AGT activity and protein. In all of these latter patients so far examined it appears that the disease is due, at least in part, to a unique protein sorting defect in which AGT is erroneously targeted to the mitochondrion instead of its normal intracellular location, the peroxisome (4). In an attempt to explain the molecular basis of this protein mistargeting phenomenon, we recently cloned and sequenced AGT cDNA from the liver of a PH1 patient with mitochondrial AGT (mAGT) and compared this with the sequence of normal human liver AGT cDNA (5, 6). This identified the presence of three point mutations, each specifying an amino acid substitution, which were subsequently shown to be common to all PH1 patients with mAGT analyzed to date (6). One of these substitutions, a proline-toleucine at residue 11 of the AGT protein (Pro-11-...

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT). The disease is notable for its extensive heterogeneity at the clinical, biochemical, enzymic and molecular genetic levels. A study of 116 PH1 patients over the past 8 years has revealed four main enzymic phenotypes: (1) absence of both AGT catalytic activity and immunoreactive AGT protein (approximately 40% of patients); (2) absence of AGT catalytic activity but presence of immunoreactive protein (approximately 16% of patients); (3) presence of both AGT catalytic activity and immunoreactive protein (approximately 41% of patients), in most of which cases the AGT is mistargeted to the mitochondria instead of the peroxisomes; and (4) a variation of the mistargeting phenotype in which AGT is equally distributed between peroxisomes and mitochondria, but in which that in the peroxisomes is aggregated into matrical core-like structures (approximately 3% of patients). Various point mutations, all occurring at conserved positions in the coding regions of the AGT gene, have been identified in these patients. The five mutations discussed in the present study, which have been found in individuals manifesting all of the four major enzymic phenotypes, account for the expressed alleles in about half of all Caucasian PH1 patients. The most common mutation found so far leads to a Gly170-->Arg amino acid substitution. This mutation, in combination with a normally occurring Pro11-->Leu polymorphism, appears to be responsible for the unprecedented peroxisome-to-mitochondrion mistargeting phenotype.

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We report here the identification of a duplication within the first intron of the gene encoding human alanine:glyoxylate aminotransferase (AGT); this duplication is closely linked to two point mutations associated with peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1 (PH1) patients. Polymerase chain reaction amplification of regions of the AGT gene including the insertion site from individuals heterozygous for this duplication, produces allele-specific fragments of different sizes. We have taken advantage of this to identify a nonsense mutation within a non-expressed allele of a compound heterozygote PH1 patient with mitochondrial AGT.

administered. Ligation of the vasa efferentia and depletion of epididymal spermatozoa decreases the amount of [32P]phosphate incorporated into both the lecithin and glycerylphosphorylcholine. We thank the Atomic Energy Commission of the United States and the International Atomic Energy Agency for a grant for the support of T.W.S. and for the purchase of equipment and isotopes.