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J. Biol. Chem., Vol. 277, Issue 3, 2336-2344, January 18, 2002
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From the Department of Biology, University College London,
Gower Street, London WC1E 6BT, United Kingdom
Received for publication, July 25, 2001, and in revised form, October 30, 2001
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.
The intermediary metabolic enzyme alanine:glyoxylate
aminotransferase 1 (AGT,1 EC
2.6.1.44) is unusual in that it is targeted to different organelles in
different mammalian species (1, 2). Although there are a number of
exceptions, there appears to be a general rule that AGT in the
hepatocytes of carnivores or insectivores (e.g. cat, dog,
shrew, mole, and hedgehog) tends to be mainly mitochondrial, whereas
that in herbivores (e.g. gorilla, orangutan, saki monkey,
rabbit, guinea pig, fruit bat, koala, and wallaby) tends to be
peroxisomal. In omnivores (e.g. marmosets, tamarins, rodents, and opossum) AGT is usually more evenly distributed between mitochondria and peroxisomes. The guinea pig has significant levels of
cytosolic AGT in addition to peroxisomal AGT (3). No mammals have been
identified so far with both mitochondrial and cytosolic AGT. However,
such a distribution has been found in the common frog (1). We have
suggested previously (1, 2) that the variable dual distribution of
organellar AGT in mammalian hepatocytes reflects a dual metabolic role
of gluconeogenesis (in the mitochondria) and glyoxylate detoxification
(in the peroxisomes). Whether cytosolic AGT, when present, has any
particular metabolic function is unknown. Gluconeogenesis might be
expected to be more important in evolutionary terms for carnivores and
insectivores because of their high protein and low carbohydrate diets,
whereas glyoxylate detoxification might be expected to be more
important for herbivores because their diets are more likely to contain
relatively high levels of oxalate and oxalate precursors, most of which
are metabolized to oxalate via glyoxylate. Oxalate cannot be
metabolized further in mammals, and when present above certain levels
oxalate causes severe problems due to the very low solubility of its
calcium salt, which can crystallize out in the kidney and urinary tract as stones (4-6). Humans seem to be an exception to the general diet-AGT distribution rule by having peroxisomal AGT (7), even though
most people could probably be categorized as omnivores.
In all the species studied so far (i.e. human, marmoset,
cat, rat, guinea pig, and rabbit), AGT is encoded by a single gene that
has the potential to encode N-terminal mitochondrial and C-terminal
peroxisomal targeting sequences (3, 8-11). 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) (see Fig.
9A). Transcription and translation from the more 5'-sites
yield a polypeptide possessing an MTS, whereas in the polypeptide
produced following transcription or translation from the more 3'-sites,
the MTS is missing.
Whereas the mitochondrial targeting of AGT appears to be fairly
straightforward, its peroxisomal targeting is rather unusual. Most
peroxisomal proteins are targeted by a C-terminal tripeptide based on
the consensus motif S/A/C-K/R/H-L/M (13, 14). This type 1 peroxisomal targeting sequence (PTS1) interacts directly with the
tetratricopeptide repeat domain of the recycling PTS1 import receptor
Pex5p (15, 16). Although the peroxisomal import of AGT is also mediated
by Pex5p (17), and is therefore presumably imported via the PTS1
pathway, its C-terminal tripeptides in mammals rarely achieve better
than a two out of three match with the consensus PTS1. At least in the
case of human AGT, its nonconsensus PTS1 (i.e. KKL) is
necessary for peroxisomal import but insufficient to direct the
peroxisomal import of a variety of reporter proteins (17, 18).
The importance of hepatic AGT in minimizing the endogenous oxalate
production in at least some mammals is clearly shown by the autosomal
recessive disorder of glyoxylate metabolism primary hyperoxaluria type
1 (PH1) (4), a potentially lethal condition in which AGT deficiency
leads to excessive oxalate synthesis and excretion and the deposition
of insoluble calcium oxalate in the kidney. PH1 also shows the
importance of the correct intracellular compartmentalization of AGT.
Although most patients have a complete absence of AGT, a significant
subset do have catalytically active AGT, but it is mistargeted from the
peroxisomes to the mitochondria (19) where it is unable to detoxify
glyoxylate efficiently, thus allowing more to be oxidized to oxalate.
It is quite common to find peroxisomal enzymes in the cytosol,
especially the classical peroxisomal marker catalase (20, 21). In the
case of guinea pig AGT, its presence in the cytosol appears to be due
to an intrinsic inefficiency with which the guinea pig peroxisomal
import machinery can deal with the nonconsensus PTS1 HRL (3). However,
it is rare to find mitochondrial enzymes in the cytosol to any
significant extent, presumably because a failure of mitochondrial
import leads to rapid degradation. The few examples that are known
result from alternative transcription or translation initiation,
similar to that found for mammalian AGT, or from import being partially
thwarted due to the presence of tightly folded import-incompetent
conformations in the protein cargo (22).
In the present study, we have attempted to gain a better understanding
of the molecular evolution of AGT targeting by studying its
distribution in a phylum not studied before (i.e. the
Amphibia). We have determined the molecular basis for its dual
mitochondrial and cytosolic localization in the liver of an amphibian
exemplar, Xenopus laevis. In addition, we have embarked on
preliminary studies that might help explain some of the unusual
characteristics of human AGT peroxisomal targeting.
Animals--
Fresh liver samples from common frog (Rana
temporis), bullfrog (Rana catesbeiana), palmate newt
(Triturus helvetica), pobblebonk (Limnodynastes
dumerillii), and Xenopus (X. laevis) were
kindly provided by Dr. Andrew Cunningham, Institute of Zoology, London, UK, or the Biological Services Department, University College London, UK.
Subcellular Distribution of AGT--
Fresh samples of liver were
fixed in 1% glutaraldehyde in 100 mM phosphate buffer, pH
7.4, and prepared for post-embedding protein A-gold immunoelectron
microscopy as described previously (7, 19, 23). Immunoreactive AGT was
detected using monospecific rabbit anti-human AGT antiserum and protein
A-gold (10 or 20 nm). In some cases, the sections were also
labeled with rabbit anti-human liver catalase antiserum and protein
A-gold (10 nm) to label the peroxisomes.
Isolation of Xenopus AGT cDNA Clones--
Standard
recombinant DNA methodologies (24) were used unless indicated
otherwise. The primers used to make the various Xenopus AGT
cDNA clones are described in Table I,
and the overall cloning strategy is outlined in Fig.
1. A total of 300,000 plaques from a
Xenopus liver cDNA expression library in the Uni-ZAP XR
vector (Stratagene) were screened using rabbit anti-human AGT
antiserum. Following secondary and tertiary screens, seven positive
plaques were identified, one of which (pXS) had a strong similarity to AGT cDNAs isolated from other species. For expression studies, pXS
was cloned into pcDNA3 (Invitrogen) to give
pXlAGT(short).
On comparison with AGTs from other species, it was clear that, although
the library clone pXS extended to the poly(A) tail at the 3'-end, at
the 5'-end it extended to only 23 bp upstream of the more 3' of the
ancestral translation start sites. Therefore, it did not include the
region homologous to the more 5'-translation start site (see Fig. 1).
To acquire more 5'-sequence, especially to determine the presence or
absence of this 5'-site, rapid amplification of cDNA ends (RACE)
was carried out (25). This was performed using the 5'/3'-RACE kit
(Roche Molecular Biochemicals) according to the manufacturer's
instructions. First strand cDNA synthesis was carried out using
Xenopus liver total RNA and primer P9 (Table I and Fig. 1).
The cDNA was purified using the High Pure PCR Product Purification
kit (Roche Molecular Biochemicals), according to the manufacturer's
instructions. After tailing of the 5'-end of the cDNA with dATP,
the dA-tailed cDNA was used as template in a PCR amplification
reaction using an oligo(dT)-anchor primer and the
Xenopus-specific primer P8. A twentieth of this product was
used as template in a second round of PCR amplification using the
PCR-anchor primer and Xenopus-specific primer P7 (Table I and Fig. 1). This yielded two products of ~70 and 350 bp that were
cloned into pGEM-T Easy (Promega). The shorter RACE product did not
extend significantly beyond the 5'-end of pXS and was termed RACE-S.
However, the longer product extended 276 bp upstream of pXS and
included the more 5'-ancestral translation start site. This RACE
product was termed RACE-L.
To check that RACE-L and pXS were derived from the same transcript,
reverse transcriptase-PCR was carried out on total Xenopus liver RNA, using oligo(dT) as the primer for first strand synthesis and
then primer pairs P1/P2 and P3/P2. The longer P1/P2 product (Xall)
appeared to be recombinogenic and refractory to cloning. The shorter
P3/P2 product (XL) was cloned into pcDNA3 to give pXlAGT(long). All clones (or PCR products in the
case of Xall) were sequenced in both strands, and where they overlapped
(see Fig. 1) the sequences were identical.
Transcript Mapping--
The RACE results could indicate that
Xenopus liver contains two types of AGT transcript, one
extending upstream of the 3'-ancestral start site but downstream of the
5'-site, and one upstream of both sites, as has been found before in
mammals such as the marmoset and rat. To test this, Xenopus
liver AGT transcripts were mapped by RNase protection. A 5'-clone was
made by digesting pXlAGT(long) with
EcoRV and XhoI, blunt ending and re-ligation to
give pXL-5' (see Fig. 1). Following linearization with
HindIII, an antisense riboprobe was generated from the T7
RNA polymerase promoter in the presence of [ N-terminal and C-terminal Constructs--
To test formally for
the presence of mitochondrial and peroxisomal targeting sequences in
Xenopus AGT, various Xenopus/human AGT and
Xenopus AGT/green fluorescent protein (GFP) constructs were
made. By using pXlAGT(long) as template,
Xenopus AGT cDNA was amplified by PCR using primer pairs
P4/P10, P4/P11, and P3/P11 (Table I and Fig. 1). The products were
digested with EcoRI and ApaI and ligated into
previously digested pcDNA3 to make clones
pXlAGT(short)-SKL,
pXlAGT(short)-KKL, and
pXlAGT(long)-KKL, respectively.
A region of pXlAGT(long) was amplified by PCR
using primers P3 and P5 (Table I and Fig. 1). The product was digested
with EcoRI and BamHI and ligated into previously
digested pEGFP (CLONTECH) to make
pXlAGT(MTS)-GFP.
To make pHsAGT-KKM, human AGT cDNA (8) was amplified
using the human-specific primer P6 and the mutagenic primer P12. The PCR product was cloned into pGEM-T Easy and cut with ApaI
and BamHI. The digested product was ligated back into the
original human cDNA clone, replacing the original region. The whole
insert was cut out of pBluescript with HindIII and
BamHI and cloned into pcDNA3 to form clone
pHsAGT-KKM.
Expression in COS Cells and Immunofluorescence
Microscopy--
SV40-transformed monkey kidney cells (COS-1), grown on
coverslips in Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal calf serum at 37 °C under 5%
CO2, were transfected with various cDNA constructs
using Superfect (Qiagen), according to the manufacturer's
instructions. Two days after transfection, the cells were washed in
phosphate-buffered saline and fixed in freshly prepared 3% (w/v)
paraformaldehyde for 15 min at room temperature, followed by
permeabilization with 1% Triton X-100 for 15 min. The cells were then
processed for immunofluorescence microscopy as described previously
(12), using mono-specific rabbit anti-human AGT antiserum and
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) or
guinea pig anti-human catalase followed by biotinylated anti-guinea pig
IgG and avidin Texas Red (Vector Laboratories). Sometimes the cells
were stained with MitoTracker (Molecular Probes) before fixation. The
coverslips were mounted onto glass slides in Mowiol (Harlow Chemical
Co. Ltd.) containing diazabicyclo-(2,2,2)octane (Sigma). The
fluorescent staining pattern was visualized in a Bio-Rad MRC1000
confocal laser-scanning microscope, and the images were digitally
recorded. A description of all the constructs using for expression
studies can be found in Table II.
AGT Is Both Mitochondrial and Cytosolic, but Not Peroxisomal, in
Amphibian Liver Cells--
The subcellular distribution of
immunoreactive AGT was determined in Xenopus, pobblebonk,
bullfrog, and common newt livers obtained from both adults and
tadpoles. As we have found previously in the case of the common frog
(1), AGT in amphibian livers appears to be both mitochondrial and
cytosolic (Fig. 2). There was no evidence
of any peroxisomal labeling. Insofar as these five species are
representative of their phylum, amphibians appear to have a
distribution of AGT that is distinct from that found in any mammal so
far studied. Interestingly, the distribution of AGT in the only reptile
to have been studied to date (i.e. the pond terrapin) shows
it also to be mitochondrial and
cytosolic.2 The presence of
mitochondrial, but not peroxisomal, AGT is compatible with the
carnivorous diets (e.g. insects, slugs, worms etc.) of adult, if not larval, amphibians and suggests a gluconeogenic, rather
than glyoxylate detoxification, role for the enzyme.
Xenopus AGT Has a High Sequence Similarity to Its Mammalian
Homologues--
The composite Xenopus AGT cDNA sequence
(GenBankTM AJ278065) shown in Fig.
3 contains an open reading frame that
encodes a protein of 415 amino acids. Two in-frame potential
translation initiation sites can be identified 24 codons apart. The
polypeptide initiating at the more 3'-site is predicted to contain 391 amino acids (one less than found in the equivalent region in AGTs from mammalian sources) and is 66% identical and 75% similar to human AGT.
The first 24 amino acids (from the more 5'-initiation site) contains
basic, hydroxyl, and hydrophobic amino acids and is deficient in acidic
amino acids, as is typical of MTSs. Almost no primary sequence identity
between this putative MTS and the 22-residue MTS in AGT of mammals,
such as marmoset, cat, and rat, could be discerned (Fig.
4). On the other hand, the C-terminal
region of Xenopus AGT is well conserved when compared with
mammalian AGTs, except that the C-terminal tripeptide in
Xenopus AGT is KKM, compared with KKL, SQL, NKL, and HRL
found in various mammalian AGTs (Fig. 4).
The Xenopus AGT Gene Encodes a Long Transcript That Includes the
MTS within the Open Reading Frame and a Short Transcript from Which It
Is Excluded--
RNase protection analysis generated two fragments
(Fig. 5), the 5'-ends of which mapped to
~39 and ~89 bp downstream of the 5'-end of
pXlAGT(long). This indicates that the
Xenopus AGT gene encodes at least two different kinds of
transcript, the longer including both of the putative translation
initiation sites and the putative MTS in its open reading frame and the
shorter excluding the more 5'-site and the MTS. Although the 5'-ends of
the long and short transcripts determined by RNase protection and RACE
do not coincide, the functional consequences as far as the nature of
the polypeptides synthesized are the same.
The subcellular localizations of the long and short forms of
Xenopus AGT can be seen following their expression in COS
cells. XlAGT(long) is targeted to mitochondria,
whereas pXlAGT(short) remains cytosolic (Fig.
6). Thus the dual mitochondrial and
cytosolic distribution of AGT in the liver cells of Xenopus,
and probably other amphibians, can be explained by alternative
transcription initiation and the production of both long and short
transcripts which encode polypeptides that either do or do not possess
an MTS.
The N-terminal 24 Amino Acids of Xenopus AGT Are Necessary for
Targeting to Mitochondria and Sufficient for the Mitochondrial
Targeting of a GFP Fusion Protein--
The observation that
XlAGT(long) is mitochondrial and
XlAGT(short) is cytosolic clearly shows the
necessity of the N-terminal 24 amino acids for the mitochondrial
targeting of Xenopus AGT. To test its sufficiency, a
construct (pXlAGT(MTS)-GFP) was made in which
this region was fused to the N terminus of GFP. GFP contains no
targeting information and when expressed in COS cells remains
cytosolic. However, when pXlAGT(MTS)-GFP was
expressed it was localized to the mitochondria, although some diffuse
labeling was also apparent (Fig. 7,
A and B). This result strongly suggests that the
N-terminal 24 amino acids of XlAGT(long) is its
MTS, although other regions of the protein might also be involved in
improving its efficiency.
The C-terminal Tripeptide of Xenopus AGT Is Not Sufficient to
Direct the Peroxisomal Targeting of Human AGT--
Unlike AGT in
mammalian livers, there is no evidence that AGT is at all peroxisomal
in amphibian livers. However, it is very difficult to provide
definitive proof against the presence of a protein in an intracellular
compartment. Peroxisomal targeting of mammalian AGTs is, at least in
part, dependent on their C-terminal tripeptides that seem to function
as rather atypical type 1 peroxisomal targeting sequences (PTS1s). In
an attempt to gain further insights into whether Xenopus AGT
has any potential for peroxisomal targeting, we have functionally
characterized its C terminus further.
Unlike the N-terminal 24 amino acid MTS of
XlAGT(long), the C terminus of XlAGT
is very well conserved with 16 of the last 20 residues being found at
the same position in the AGTs from at least one mammal (see Fig. 4). In
all mammals studied so far, AGT is at least partly peroxisomal even
though the tripeptides at the C termini are highly variable
(i.e. KKL in human and marmoset (8, 9), NKL in cat and rat
(11, 26), HRL in guinea pig (3), and SQL in rabbit (9)). In no cases
are these better than a two out of three match to the consensus PTS1 of
S/A/C-K/R/H-L/M (13, 14). The C-terminal tripeptide of
Xenopus AGT is KKM which is also a two out of three match
and rather similar to the C terminus of human AGT. The nonperoxisomal
distribution of both XlAGT(long) and
XlAGT(short) clearly shows that KKM cannot
direct the peroxisomal targeting of Xenopus AGT. To test
whether it was able to target a mammalian AGT to peroxisomes, a
construct was made in which the C-terminal KKL of human AGT was
replaced by the C-terminal tripeptide of Xenopus AGT
(i.e. KKM). When this construct (pHsAGT-KKM) was
expressed in COS cells the distribution was cytosolic, no peroxisomal
labeling was evident (Fig. 7, E and F), showing
that KKM is not capable of directing either human or Xenopus
AGT to peroxisomes.
The Consensus PTS1 SKL Is Able to Direct the Peroxisomal Targeting
of Xenopus AGT but the Nonconsensus PTS1 of Human AGT Is Not--
To
check that XlAGT did not contain any sequence or structural
elements incompatible with peroxisomal targeting and import, the
C-terminal tripeptide of XlAGT(short) was
replaced by the canonical PTS1 SKL. When this construct
(pXlAGT(short)-SKL) was expressed in COS cells
its distribution was peroxisomal (Fig. 7, G and
H), indicating that such inhibitory elements were not present.
The C-terminal tripeptide of human AGT, KKL, is necessary for its
peroxisomal targeting but insufficient to direct the peroxisomal import
of reporter proteins. One possible explanation for this is that human
AGT contains additional targeting information that makes KKL acceptable
as a PTS1. XlAGT is 66% identical and 75% similar to
HsAGT and therefore might also contain this putative extra targeting information even though it is not targeted to peroxisomes. To test this hypothesis, constructs were made in which the
C-terminal KKM of Xenopus AGT was replaced by KKL. When either pXlAGT(short)-KKL or
pXlAGT(long)-KKL was expressed in COS cells, no
peroxisomal labeling was detectable. XlAGT(short)-KKL was cytosolic and
XlAGT(long)-KKL was mitochondrial (Fig.
8). Therefore, whatever is present in
HsAGT that enables KKL to target it to peroxisomes, it
appears to be absent from XlAGT.
Evolution of AGT Targeting in Amphibians--
The dual
mitochondrial and cytosolic localization of AGT in Xenopus
liver can be explained by the use of alternative transcription start
sites such that the region encoding an N-terminal MTS is either
included or excluded from the open reading frame (see Fig. 9). Such a mechanism has also been shown
to be responsible for the mitochondrial and cytosolic distribution of a
number of other enzymes, such as the products of the HTS1,
VAS1, MOD5, CCA1 and LEU4
genes in Saccharomyces cerevisiae (22). Alternative
transcription initiation also explains the rather different
mitochondrial and peroxisomal distribution of AGT in some mammals, such
as the marmoset and rat (9, 10, 12). The absence of peroxisomal AGT in Xenopus liver can be attributed to the absence of a PTS1.
However, it is not clear why the C terminus of Xenopus AGT
should be so well conserved and especially why the C-terminal
tripeptide should be so similar to that of human and marmoset AGT
(i.e. KKM compared with KKL). Although amphibian AGT genes
other than that of Xenopus have not been sequenced, it is
highly probable that the same molecular basis for dual mitochondrial
and cytosolic distribution of AGT also occurs in common frog,
pobblebonk, bullfrog, and common newt.
The 5'-structure of the Xenopus AGT gene and its ability to
produce two transcripts would suggest that dual AGT
compartmentalization was present early in the evolution of vertebrates.
Although the lack of any conservation between the Xenopus
and mammalian AGT MTSs might suggest that mitochondrial targeting of
AGT in amphibians and mammals was acquired separately, it is perhaps
more likely that mitochondrial targeting was present in a common
ancestor and that lack of conservation simply reflects the well
recognized primary sequence degeneracy of MTSs (27). What is much less clear is whether the nonmitochondrial AGT in early vertebrates was
cytosolic or peroxisomal.
Unlike other peroxisomal enzymes, the C termini of AGT in different
mammalian species are highly variable. For example, they are KKL in
human and marmoset, NKL in cat and rat, SQL in rabbit, and HRL in
guinea pig. In all cases these tripeptides can direct the peroxisomal
import of the protein, but none fit the conservative consensus PTS1
(see Introduction). The only mammalian species identified so far in
which such a PTS1 is present is in bovine AGT (GenBankTM
accession number BE750720), the C terminus of which is SKL. AGT
cDNAs have been isolated in a number of invertebrates and higher
plants. In Drosophila melanogaster (GenBankTM
accession number CAA58024) and Caenorhabditis elegans (GenBankTM accession number Q94055) the C terminus of AGT
is SKI, and in Arabidopsis thaliana (GenBankTM
accession number AAD28669) (28) and Fritilliaria agrestis (GenBankTM accession number AAB95218) it is SRI. AGT is
peroxisomal in A. thaliana (28) and is predicted to be
peroxisomal in the other species as well. Amphibian AGT is the only
naturally occurring form of AGT shown not to be able to be targeted to
peroxisomes, and thus KKM is the only naturally occurring AGT
C-terminal tripeptide that is unable to act as a PTS1, even of a
nonconsensus variety. From the foregoing information, it is impossible
to choose between two different scenarios for the evolution of AGT
targeting in early vertebrate evolution. First, AGT could have been
mitochondrial and peroxisomal, with the latter being lost in amphibians
due to mutation of the C-terminal tripeptide. Second, AGT could have been mitochondrial and cytosolic, peroxisomal targeting having been
acquired in mammals at the expense of cytosolic localization. In the
latter case, peroxisomal targeting in mammals would have had to have
been acquired separately from that in invertebrates and higher plants.
The similarity between the C-terminal tripeptides of Xenopus
AGT (i.e. KKM) and human/marmoset AGT (i.e. KKL)
is difficult to understand unless either peroxisomal targeting has only
recently been lost in the evolutionary history of amphibians or the C
terminus of AGT contributes to some other essential characteristic of
the protein. As peroxisomal AGT targeting was probably lost before the
divergence of Xenopus, common frog, bullfrog, pobblebonk, and common newt, the former possibility seems unlikely. As far as the
latter possibility is concerned, it is interesting to note that the
C-terminal 21 amino acids of Xenopus AGT are 81% identical to human AGT, which is much higher than the average for the whole molecule (i.e. 66%). Pressure for such sequence
conservation could come from the requirement to maintain correct
folding and catalytic activity. Whether this is actually the case is
not currently known, but it is relevant to note that human AGT still
dimerizes when the C-terminal KKL is deleted (29) and is still
catalytically active when a C-terminal His tag is attached (30).
Xenopus AGT Lacks the Ancillary Peroxisomal Targeting Information
Predicted to Be Present in Human AGT--
The peroxisomal targeting of
mammalian AGTs is far from being fully understood. Although it is
dependent on the presence of the PTS1 import receptor Pex5p, but not
the PTS2 import receptor Pex7p (17), the C-terminal tripeptides found
in different species are highly variable and in most cases do not fit
the PTS1 consensus sequence of S/A/C-K/R/H/-L/M.
The peroxisomal targeting of human AGT has been studied in more detail
than that in the other species. Although the C-terminal KKL is
necessary for the peroxisomal targeting of human AGT, it is
insufficient to target reporter proteins such as chloramphenicol acetyltransferase, firefly luciferase, and GFP to peroxisomes (17, 18).
The unusual nature of the PTS1 in human AGT has been confirmed recently
when it was shown that AGT does not interact with human Pex5p in the
yeast two-hybrid system, cannot compete with the peroxisomal import of
other PTS1 proteins, and its peroxisomal import can be inhibited by
overexpression of Pex5p (31). In an attempt to explain this atypical
behavior, we have suggested that human AGT might possess ancillary
peroxisomal targeting information other than at the C terminus and that
interaction with Pex5p might require the presence of a an additional
adaptor molecule (31).
The observation that Xenopus AGT cannot be targeted to the
peroxisomes, despite its high similarity to human AGT, appears to be
due to the fact that KKM cannot act even as a nonconsensus PTS1. In
addition, the combined failure of KKM to direct the peroxisomal targeting of human AGT and KKL to direct the peroxisomal targeting of
Xenopus AGT strongly suggests that Xenopus AGT
does not contain the ancillary peroxisomal targeting information
predicted to be present in human AGT. The significance of this for our
understanding of the evolution of AGT targeting remains to be seen.
Nevertheless, the sequence similarity between Xenopus and
human AGT opens up the possibility that identification of this
ancillary targeting information might be possible by studying the
ability of various human-Xenopus chimeric AGTs to target to peroxisomes.
Whatever the results of such studies, they are likely to
add yet another chapter to the remarkable story of the evolution of AGT
targeting, which so far includes the following: (a) the repeated loss of one or other of the alternative transcription and
translation initiation sites in mammals with the inclusion or exclusion
of the MTS from the open reading frame (12); (b) the
positive selection for loss or decreased efficiency of the MTS in
primates (32); (c) the generation and high population frequency of a cryptic MTS in humans (33, 34); (d) the
manifestation of this cryptic MTS in patients with a lethal hereditary
disease resulting in peroxisome-to-mitochondrion mistargeting (30,
34).
We thank Dr. Andrew Cunningham (Institute of
Zoology, London, UK) for providing some of the liver samples and
Dr. Imelda Gallagher for carrying out the immunoelectron microscopy.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ278065.
§
To whom correspondence should be addressed: MRC Laboratory
for Molecular Cell Biology, University College London, Gower St., London WC1E 6BT, UK. E-mail: c.danpure@ucl.ac.uk.
Published, JBC Papers in Press, November 2, 2001, DOI 10.1074/jbc.M107047200
2
P. Fryer and C. J. Danpure, unpublished observations.
The abbreviations used are:
AGT, alanine:glyoxylate aminotransferase 1;
GFP, green fluorescent protein;
MTS, mitochondrial targeting sequence;
PTS1, peroxisomal targeting
sequence type 1;
RACE, rapid amplification of cDNA ends.
Molecular Basis for the Dual Mitochondrial and Cytosolic
Localization of Alanine:Glyoxylate Aminotransferase in Amphibian Liver
Cells*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used in the present study
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Fig. 1.
Schematic diagram of Xenopus
AGT cloning strategy. Full-length Xenopus AGT
cDNA is represented at the top of the diagram with the
box indicating the open reading frame. The relative
positions to which the oligonucleotide primers (P1-P5 and
P7-P12) map are indicated. The primer sequences are shown
in Table I, and their uses are explained in the text. Representations
of the PCR products and clones are aligned approximately to their
position on the full-length cDNA. 
, Xall was not cloned.
MTS, region encoding the putative mitochondrial targeting
sequence. TL1 and TL2, positions of the two
ancestral translation start sites. TGA, position of stop
codon. The position of the EcoRV site referred to in the
text is also indicated. The diagram is not drawn to scale.
-32P]UTP
(600 Ci/mmol). Total RNA (20 µg) prepared from Xenopus
liver was hybridized for 16 h at 45 °C with the antisense
riboprobe (105 cpm). Following digestion with RNase A and
T1, the protected fragments were separated on a 6% denaturing
polyacrylamide sequencing gel and visualized by autoradiography using
Biomax MR-1 film (Eastman Kodak Co.).
Description of the various AGT constructs used in the present study and
their subcellular distribution in transfected COS cells
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Subcellular distribution of AGT in amphibian
liver cells. The panels show sections of liver obtained
from pobblebonk tadpole (A), common frog adult
(B), Xenopus adult (C), and bullfrog
tadpole (D) labeled for immunoreactive AGT using
post-embedding protein A-gold (10 or 20 nm) immunoelectron microscopy.
Peroxisomes are indicated by arrows; cytosolic labeling is
indicated by arrowheads (in C only). A study of
numerous images showed that, in all cases, labeling was mitochondrial
and cytosolic. No peroxisomal labeling was evident. Adult bullfrog,
common frog tadpole, adult pobblebonk, Xenopus tadpole, and
palmate newt adult and tadpole gave similar results. Bars,
0.5 µm.
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Fig. 3.
Nucleotide and amino acid sequence of
Xenopus AGT. The cDNA sequence shown is a
composite of Xall and the 3'-end of pXS. The predicted amino acid
sequence is shown beneath the first base of every codon. The two
ancestral translation start sites are double underlined. The
polyadenylation signal in the 3'-untranslated region is
underlined as are the stop codons in the 5'-untranslated
region. The putative pyridoxal phosphate-binding site is
boxed. Triangles mark the 5'-ends of pXL and pXS.
This sequence has been deposited in the GenBankTM DNA
(accession number AJ278065).
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Fig. 4.
N-terminal and C-terminal amino acid
alignments. The predicted N- and C-terminal amino acid sequences
of Xenopus AGT are aligned with the homologous regions of
various mammalian AGTs. The positions of the ancestral translation
start sites 1 and 2 are shaded, and the initiating
methionines are in bold and underlined. The
C-terminal tripeptides are in bold. *, residues conserved in
mammals if contained within the open reading frame. +, residues in
XlAGT that are found in at least one mammalian AGT. Unlike
the C terminus, which is very well conserved between species, the
N-terminal leader sequence is very poorly conserved.
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Fig. 5.
Transcript analysis of Xenopus
AGT by RNase protection. A shows the
autoradiograph resulting from the RNase protection assay. B
shows the relative sizes of the antisense riboprobe and the protected
fragments compared with various XlAGT clones. The
numbers indicate the estimated sizes of the protected
fragments in nucleotides (i.e. ~259 and ~209) compared
with the size of the probe (i.e. 298). A, the
heavily overloaded self-hybridization riboprobe tract (pXL-5') showed
that although tiny amounts of lower sized fragments were present, they
did not co-electrophorese with the protected bands. No bands were
detected in the tRNA (20 µg) tract. The tract containing 20 µg of
total Xenopus liver RNA gave two protected bands.
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Fig. 6.
Intracellular distribution of the long and
short forms of Xenopus AGT in transfected COS
cells. COS cells were transfected with either
pXlAGT(long) (A-D) or
pXlAGT(short) (E-H). Cells were
double-labeled for AGT (A, C, E, and G) and
either the peroxisomal marker catalase (B and F)
or the mitochondrial marker MitoTracker (D and
H). XlAGT(long) co-localized with
MitoTracker, but not catalase. XlAGT(short) was
diffusely distributed throughout the cell with no evidence of
co-localization with either catalase of MitoTracker. Bar, 10 µm.
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Fig. 7.
Intracellular distribution of various
Xenopus and human fusion proteins. COS cells were
transfected with either pXlAGT(MTS)-GFP
(A and B), pHsAGT (C and
D), pHsAGT-KKM (E and F),
or pXlAGT(short)-SKL (G and
H). Cells were double-labeled for GFP autofluorescence
(A) or AGT (C, E and G) and
MitoTracker (B) or catalase (D, F and
H). XlAGT(MTS)-GFP co-localized
mainly with MitoTracker (A and B) but with some
diffuse staining also. HsAGT co-localized with the
peroxisomal marker catalase (C and D), but when
its C-terminal KKL is replaced by KKM it was diffusely distributed with
no catalase co-localization (E and F). Unlike
pXlAGT(short), which was diffusely distributed
(see Fig. 6, E-H), pXlAGT(short)-SKL
co-localized with the peroxisomal marker catalase (G and
H). Bar, 10 µm.
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Fig. 8.
Intracellular distribution of short and long
Xenopus AGT in which the C-terminal KKM has been
replaced by KKL. COS cells were transfected with either
pXlAGT(short)-KKL (A and
B) or pXlAGT(long)-KKL
(C-F). Cells were double-labeled for AGT (A, C,
and E) and either catalase (B and D)
or MitoTracker (F). XlAGT(short)-KKL
was diffusely distributed with no evidence for co-localization with
catalase. XlAGT(long)-KKL was co-localized with
MitoTracker, again with no evidence of any co-localization with
catalase. Bar, 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Alternative transcription and translation
initiation and the dual compartmentalization of AGT. A,
diagrammatic structure of the archetypal AGT gene. A and
B, the two ancestral transcription start sites. 1 and 2, the two ancestral translation start sites.
MTS, region with potential to encode an N-terminal
mitochondrial targeting sequence. PTS1, region with
potential to encode a nonconsensus PTS1 in some mammalian species.
B, presence (+) or absence ( 
) of transcription start sites
A and B and translation start sites 1 and 2 in
AGT from various species, together with the presence or absence of a
C-terminal PTS1. Subcellular distribution: MITO,
mitochondrial; PEROX, peroxisomal; CYTO,
cytosolic (case represents the approximate proportional distribution).
1 This site is presumed to be present due to an
appropriate cDNA being isolated (8) but not detected by primer
extension (M. Sato, S. Toné, T. Ishikawa, P. E. Purdue,
C. J. Danpure, and Y. Minatogawa, submitted for publication).
2 This site is shown to be predominant by primer extension
(M. Sato, S. Toné, T. Ishikawa, P. E. Purdue, C. J. Danpure, and Y. Minatogawa, submitted for publication).
3 This site is presumed to be present due to an
appropriate cDNA having been isolated (9).
4 Transcription from this site is induced by gluconeogenic
stimuli (35). 5 Distribution of AGT in rat liver is highly
variable depending on diet and presence of gluconeogenic stimuli
(36).
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a Medical Research Council (MRC) Ph.D. studentship.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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