Molecular Basis for the Dual Mitochondrial and Cytosolic Localization of Alanine:Glyoxylate Aminotransferase in Amphibian Liver Cells

Christopher J. Danpure,Joanna D. Holbrook

doi:10.1074/jbc.m107047200

Abstract

To gain further insights into the molecular basis of the evolution of alanine:glyoxylate aminotransferase (AGT) intracellular targeting in vertebrates, we have studied the molecular basis of its dual mitochondrial and cytosolic distribution in amphibian liver cells. The AGT gene in Xenopus laevis encodes a polypeptide of 415 amino acids, which includes a 24-residue N-terminal mitochondrial targeting sequence (MTS), at either end of which are located two in-frame potential translation start sites. This MTS is necessary to target Xenopus AGT and sufficient to target a green fluorescent fusion protein to mitochondria in transfected COS cells. The C-terminal tripeptide (KKM), despite being similar to the nonconsensus type 1 peroxisomal targeting sequence in human AGT (KKL), was unable to target Xenopus AGT or human AGT to peroxisomes. The Xenopus AGT gene produces two types of transcript. The longer form encodes a polypeptide that contains the MTS and is targeted to mitochondria. The shorter form encodes a polypeptide that does not contain the MTS and remains in the cytosol. These results are discussed not only in terms of the molecular evolution of AGT targeting but also in terms of the ancillary requirements for the peroxisomal targeting of human AGT.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ278065
To gain further insights into the molecular basis of the evolution of alanine:glyoxylate aminotransferase (AGT) intracellular targeting in vertebrates, we have studied the molecular basis of its dual mitochondrial and cytosolic distribution in amphibian liver cells
The variable targeting of AGT in mammals appears to be dependent on the variable use of two in-frame translation start sites, which straddle the region encoding the 22-amino acid-cleavable N-terminal mitochondrial targeting sequence (MTS), and two groups of transcription start sites, one upstream of both translation start sites and one between them [10, 12]

Summary

TABLE I Primers used in the present study

The following primers contain restriction sites (bold and underlined): P1 KpnI, P2 XhoI, P3 EcoRI, P4, EcoRI, P5 BamHI, P10 ApaI, P11 ApaI, P12 BamHI. Mapping positions are calculated from xenopus (X) or human (H) AGT cDNA

Maps to Strand

EXPERIMENTAL PROCEDURES

Mitochondrial and Cytosolic AGT in Amphibian Liver Cells

RESULTS

Peroxisomal Cytosolic Cytosolic Mitochondrial

DISCUSSION