Plant Molecular Biology 37: 99–108, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Characterization of a single soybean cDNA encoding cytosolic and
glyoxysomal isozymes of aspartate aminotransferase
Joan S. Gebhardt
, Gregory J. Wadsworth
and Benjamin F. Matthews
U.S. Department of Agriculture, Agriculture Research Service, Plant Molecular Biology Laboratory, Beltsville,
MD, 20705, USA (
author for correspondence);
Buffalo State College, Department of Biology, 1300 Elmwood
Avenue, Buffalo, NY, 14222, USA
Received 31 March 1997; accepted in revised form 3 December 1997
Key words: aspartate aminotransferase, cDNA, glyoxysome, isozyme, peroxisomal targeting signal, soybean
A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate amino-
transferase (AAT; EC 126.96.36.199) was isolated. Genomic Southern blots and analysis of genomic clones indicated
pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid
identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Align-
ment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with
characteristics of a peroxisomal targeting signal, designated PTS2, including the modiﬁed consensus sequence
-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT
cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons
produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encodedprotein
reactedwiththreesoybeanAATisozymes,AAT1(glyoxysomal),AAT2(cytosolic),and AAT3 (subcellularlocation
unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and
the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of puriﬁed
AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the
PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the
two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to
generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.
Aspartate aminotransferase (AAT; EC 188.8.131.52) cata-
lyzes the reversible transfer of the amino group of
aspartate to 2-oxoglutarate to form oxaloacetate and
glutamate. The enzyme is ubiquitous in higher euka-
ryotes and has been extensively studied because of its
central role in several metabolic processes, including
a pivotal role in carbon and nitrogen metabolism in
plants (reviewed in [13, 20, 43]).
Many plants including soybean have multiple
isozymes of AAT. Five isozymic forms of soybean
Thenucleotidesequencedatareportedwill appear intheEMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number AF034210.
AAT can be distinguished by native agarose gel elec-
trophoresis  and are designated AAT1 to AAT5.
Organelle fractionation studies have shown different
AAT isozymes are localized within distinct subcellu-
lar compartments in numerous plant species including
castor bean , cucumber , maize , and spin-
ach . In soybean, isozymes of AAT are localized in
glyoxysomes (AAT1), the cytosol (AAT2), mitochon-
dria (AAT4), and chloroplasts (AAT5) [41, 42]. The
subcellular location of AAT3 has not been determined
although it is most abundant in green tissue .
In plants AAT has been proposed to play sever-
al metabolic roles but little is known about the exact
physiological role of each isozyme. They may have a
general role in shuttling reducing equivalents, via the