123 A NEW FAMILY OF PROTEINS (rBAT AND 4F2hc) INVOLVED IN CATIONIC ...
J. exp. Biol. 196, 123–137 (1994)
123
Printed in Great Britain © The Company of Biologists Limited 1994
A NEW FAMILY OF PROTEINS (rBAT AND 4F2hc) INVOLVED
IN CATIONIC AND ZWITTERIONIC AMINO ACID
TRANSPORT: A TALE OF TWO PROTEINS IN SEARCH
OF A TRANSPORT FUNCTION
MANUEL PALACÍN
Departament de Bioquímica i Fisiologia, Facultat de Biologia, Universitat de
Barcelona, Avenuda Diagonal 645, 08028 Barcelona, Spain
Summary
The currently identified cDNA clones of mammalian amino acid transporters can be
grouped into five different families. One family is composed of the proteins rBAT and the
heavy chain (hc) of the cell surface antigen 4F2. RNAs encoding these two proteins
induce a system bo,+-like (rBAT) and a system y+L-like (4F2hc) activity in Xenopus
oocytes. Surprisingly, rBAT and 4F2hc do not seem to be pore-forming proteins. This
finding supports the hypothesis that rBAT and 4F2hc are subunits or modulators of the
corresponding amino acid transport systems. Expression of rBAT in oocytes induces
high-affinity transport of cystine, which is shared with transport of cationic and
zwitterionic amino acids. The rBAT gene is expressed mainly in kidney and small
intestine. The rBAT protein is localized to the microvilli of proximal straight tubules of
the kidney and mucosa from the small intestine. This finding is consistent with the
involvement of rBAT in a high-affinity resorption system for cystine in the proximal
straight tubule of the nephron. All of these characteristics suggest that rBAT is a good
candidate for a cystinuria gene. Cystinuria is an inheritable defect in high-affinity
transport of cystine, shared with cationic amino acids, through epithelial cells of the renal
tubule and intestinal tract. Very recently, point missense mutations have been found in
the rBAT gene of cystinuria patients. The most frequent rBAT mutation, M467T
(threonine substitution of methionine at residue 467) nearly abolished the amino acid
transport activity elicited by rBAT in oocytes. This result offers convincing evidence that
rBAT is a cystinuria gene. Biochemical, cytological and genetic approaches are now
needed to delineate the mechanism of action of rBAT and 4F2hc in the transport of amino
acids.
Introduction
Amino acid transport across the plasma membrane of mammalian cells is catalyzed by
proteins that recognise, bind and shuttle these metabolites between the extracellular and
the intracellular compartments. In the last 4 years, a rapidly growing number of cDNA
sequences encoding proteins related to amino acid plasma membrane transport have
appeared (reviews include: Bertran et al. 1994; Kanai et al. 1994; Kanner and
Kleinberger-Doron, 1994; Macleod et al. 1994). However, cDNAs encoding other
Key words: rBAT, 4F2hc, system bo,+, system y+L, amino acid transport, cystinuria
124
M. PALACI´N
significant amino acid transport activities have yet to be cloned (e.g. sodium-dependent
systems, such as NBB, Bo,+ and N, and sodium-independent systems, such as the
ubiquitous system L and anionic amino acid transporters; see the introduction to this
section in this volume by MacLeod et al. 1994). Although only a few amino acid
transporter structures are known, functionally similar transporters can already be grouped
into four different gene families (Table 1): (i) sodium-independent transporters for
cationic amino acids; CAT, (ii) amino acid transporters within the sodium- and chloride-
dependent neurotransmitter transporter superfamily, (iii) sodium-dependent, and
probably potassium-dependent and chloride-independent, anionic and zwitterionic amino
acid transporters, and (iv) sodium-dependent transporters for sugars, amino acids,
vitamins and nucleosides. In addition to these amino acid transporters, which are
predicted to have multiple transmembrane domains, two homologous proteins, rBAT
(also named D2, NAA-Tr or NBAA-Tr) and the heavy chain of the cell surface antigen
4F2 (4F2hc), which are believed to contain a single transmembrane domain, induce
amino acid transport activity in Xenopus oocytes. It has been postulated that these two
proteins may not be the actual transporters but may represent activating subunits of
multimeric transporters. These two proteins are the subject of the present review.
Cloning and identification of rBAT and 4F2hc, a new family of proteins involved in
amino acid transport
Expression-cloning resulting in amino acid transport activity in Xenopus oocytes was
used in three laboratories to isolate cDNAs encoding putative transporters from rabbit,
rat and human kidney (Bertran et al. 1992b, 1993; Lee et al. 1993; Tate et al. 1992;
Wells and Hediger, 1992). These cDNA clones encode proteins that share 80–85 %
sequence identity. The human cDNA cloned by Hediger’s group (Lee et al. 1993) lacks
66 nucleotides encoding 22 amino acids at the C terminus that are present in our human
clone and in the rat and rabbit clones (Bertran et al. 1993). For clarity, the term rBAT
will refer to human, rat and rabbit clones in this review. rBAT shares 30 % amino acid
sequence identity (50 % similarity) with another protein, the heavy chain (hc) of the
mouse and human surface antigen 4F2 (Parmacek et al. 1989; Quackenbush et al. 1987;
Teixeira et al. 1987). The cDNA encoding this protein was originally cloned in 1987 for
its ability to react with a monoclonal antibody raised against a lymphoblastic cell surface
antigen of unknown function (Quackenbush et al. 1987; Teixeira et al. 1987). Because
4F2hc shared sequence similarity with rBAT, synthetic 4F2hc RNA was tested in
Xenopus oocytes and found to induce amino acid transport activity in this system
(Bertran et al. 1992a; Wells et al. 1992). Both rBAT and 4F2hc proteins share several
structural features: both lack a leader sequence, have almost identical hydrophilicity
profiles (Fig. 1) and share four highly conserved (67–80 % identity) regions (10–18
amino acids long) in the putative extracellular domain (Fig. 2A). Both proteins contain
an extracellular cysteine residue located four amino acids from the transmembrane
region and both extracellular domains show significant homology with a family of
-amylases and -glucosidases. Interestingly, the catalytic consensus site of these
glycosidases is not conserved in rabbit rBAT or 4F2hc, which is consistent with the
Proteins involved in amino acid transport
125
− -
- and Cl+
-dependent+
12–14
12
6, 8, 10
12
1, 4
1
domains
Number of
transmembrane
1994).
et al.
cotransporters). Within this family, Kanner and−
isoforms
and Cl
isoforms
+
− AG
L
Probable related system
+ y
GABA isoforms
GABA and beta isoforms
Gly
X
ASC isoforms
A
o,+ b
+ y
(1994). The amino acid transporters within the superfamily of Na
et al.
ve families with their probable amino acid transport systems. In most cases this
− -dependent transporters (Na
rst four families share a multiple membrane-spanning model. In contrast, the family
Proteins
ve subtypes: GAT1, GAT2, GAT3 (including brain GAT3a and heart GAT3b), GAT4
- and Cl+
. The cDNA SAAT1 (sodium-dependent amino acid transporter-1) showed system A amino acid
CAT1; CAT2; CAT2a
GAT1; GAT2
GAT3; GAT4; Taut
GLYT
BGT-1; PRO
GLAST; GLT-1; EAAC-1
SATT; ASCT1
SAAT1
rBAT
4F2hc
+
Protein families related to amino acid transport in mammals
Table 1.
and countertransport of K+
− -dependent transporters
-dependent transporters+
- and Cl
- and K
-dependent transporters for sugars,
+
+
+
amino acids, vitamins and nucleosides
Family
Cationic amino acid transporters (CAT)
Na
Na
Na
Subunits of amino acid transporters
Cloned amino acid transporters in mammals have been grouped into fi
connection is not clearly established. The deduced proteins of the fi
composed by rBAT and 4F2hc (tentatively shown as subunits of amino acid transporters) contain 1–4 transmembrane domains, depending on predictions of
protein structure. Cationic amino acid transporters are reviewed by MacLeod
dependent neurotransmitter transporters are included here as Na
Kleinberger-Doron (1994) subdivided GABA transporters into fi
(including brain GAT4a, GAT4b and GAT4c) and GABA/betaine (also named BGT-1). Three high-affinity glutamate transporter cDNAs (GLAST, GLT-1 and
EAAC-1) and two highly homologous cDNAs (SATT and ASCT1), which express amino acid transport activity resembling system ASC, share sequence
homology and cotransport of Na
transport activity when expressed in COS cells and it is highly homologous to the sodium/glucose cotransporters, within the family of Na
transporters for sugars, amino acids, vitamins and nucleosides of eukaryotes and prokaryotes (reviewed in Bertran
126
M. PALACI´N
Human rBAT
4
3
2
1
0
−1
−2
−3
y
t
i
c
100
200
300
400
500
600
obi
oph
Human 4F2 hc
ydr
H
3
2
1
0
−1
−2
−3
100
200
300
400
500
Amino acid residue
Fig. 1. Hydropathy plots for human rBAT and human 4F2hc deduced proteins. The plots have
been drawn using Kyte-Doolittle’s algorithm with a window of nine amino acids (Bertran,
1993). The y-axis shows the hydrophobicity scale, and the x-axis shows the amino acid
residue. Human rBAT and 4F2hc proteins are 685 and 529 amino acids long, respectively.
Hydrophobic regions have positive y values and are considered to be potential membrane-
spanning domains if the value is higher than 2.5. Both deduced proteins (rBAT, Bertran et al.
1993; 4F2hc, Teixeira et al. 1987) have an overall similar hydropathy plot and contain a single
region (around amino acid residue 100) with a hydrophobicity value higher than 2.5.
finding that rBAT expression in Xenopus oocytes confers no -amylase or maltase
activity (Wells and Hediger, 1992).
The two proteins appear to have different functions. rBAT induces a sodium-
independent bo,+-like activity in Xenopus oocytes. The bo,+ system was first described in
mouse blastocysts (Van Winkle et al. 1988) and is a high-affinity, sodium-independent
transport system for cationic and zwitterionic amino acids, but not for cystine (L. J. Van
Winkle, personal communication). Nevertheless, rBAT induces the transport of both
cationic/zwitterionic amino acids and cystine, when expressed in the Xenopus oocytes,
with Km values in the micromolar range (Table 2). The kinetics of L-cystine transport are
shown in Fig. 3. Kinetic analysis and cross-inhibition studies provide evidence that rBAT
induces a single transport activity (Bertran et al. 1992b). This transport activity is not
present in stage VI Xenopus oocytes unless synthetic rBAT RNA is injected (Fig. 3;
Bertran et al. 1992b,c; McNamara et al. 1991). Microinjected synthetic 4F2hc RNA
Proteins involved in amino acid transport
127
A
C
L678P
C
B
T652R
C
P615T
29 %
40 %
M467T
S-S
c
4 %
Outer membrane
41 %
Plasma
membrane
R181Q
Inner membrane
27 %
c
c
Outer
N
Catalytic light
Inner
subunit
10 %
rBAT, 4F2hc
(unidentified)
N
N
rBAT
4F2hc
Fig. 2. (A) Schematic comparison of human rBAT and human 4F2hc deduced proteins.
Percentages indicate amino acid sequence identity between both proteins. Shaded areas
represent small regions of 10–18 amino acid residues of high homology (identity 67–80 %)
between both proteins. Gaps for the alignment smaller than 10 amino acid residues are not
represented or combined. The locations of cystinuria-specific missense mutations in the
protein rBAT (R181Q, arginine-188→glutamine; M467T, methionine-467→threonine;
P615T, proline-615→threonine; T652R, threonine-652→arginine; L678P, leucine-
678→proline) are indicated. Mutations R181Q and M467T (also mutation M467K;
methionine-467→lysine; which is not indicated in the figure) are located in well-conserved
regions of rBAT and 4F2hc. Interestingly, mutation P615T involves a proline residue which is
conserved in human rBAT and 4F2hc. (B) Hypothetical heterodimeric representation of rBAT
and 4F2 proteins. This diagram represents the proteins rBAT and 4F2hc (left) as type II
membrane glycoproteins, with an intracellular N terminus, a single membrane-spanning
domain (not drawn to scale) and a large extracellular domain with potential N-glycosylation
sites (four out of six potential N-glycosylation sites in human rBAT are indicated by Y) and
the C terminus. In the extracellular domain, next to the transmembrane segment a conserved
cysteine residue (c) is present in both proteins. To the right, a putative ‘catalytic light subunit’
is represented as a multiple membrane-spanning protein (i.e. a pore-forming protein). The 4F2
‘light subunit’ has been detected as a highly hydrophobic and non-glycosylated approximately
40 kDa protein after immunoprecipitation of the 4F2 complex in non-reducing conditions (see
text for details). This light subunit has not been cloned; therefore, its topological
representation here is only speculative. The sketch shows a disulfide bridge (-S–S-) between
the subunits. The putative ‘rBAT light subunit’ has not yet been identified.
elicits a transport activity quite distinct from rBAT activity (Bertran et al. 1992a; Wells et
al. 1992). It appears to enhance a pre-existing transport activity with y+-like
characteristics. Indeed, expression of 4F2hc induces sodium-dependent uptake of both L-
128
M. PALACI´N
Table 2. Kinetic variables for the rBAT-induced transport activity in oocytes
Km
Ki
Vmax
Amino acid
( mol l−1)
( mol l−1)
(pmol min−1)
Reference
L-Arginine
105
47–56
29
a
L-Lysine
75–298
a
L-Ornithine
197–222
a
L-Cystine
30–67
90–184
4–16
a,b,d,e
L-Leucine
22(r)–128
172–199
35;
97 % (r)
a,c
L-Phenylalanine
29(r)
100 % (r)
c
L-Histidine
167(r)
126 % (r)
c
L-Methionine
71(r)
151 % (r)
c
L-Alanine
800–4900
56 % (r)
a,c
L-Tryptophan
250(r)
11 % (r)
c
L-Serine*
b
L-Glutamine*
b
Citrulline*
b
L-Cysteine
†
a
L-Threonine
†
a
L-Valine
†
a
D-Ornithine
3700–7600
a
D-Lysine
†
b
Uptake values and kinetic variables refer to human, rat and rabbit rBAT-induced transport in Xenopus
oocytes. The range of kinetic variables for L-cystine transport corresponds to human, rat and rabbit
rBAT expression. No kinetic variables for amino acids other than L-cystine have been determined for
human rBAT expression.
The Ki values have been determined only for the expression of rabbit rBAT.
(r) refers to rat rBAT.
Vmax values are given as pmol min−1 oocyte−1. When given as a percentage, the values are referred to
that of Phe.
* indicates amino acids which have been shown to be carried by the expressed transport activity, but
no kinetic data are available.
† indicates those amino acids that have been shown to inhibit rBAT-induced uptake activity. rBAT
does not induce transport of L-proline, methyl-aminoisobutyric acid, L-glutamate, L-aspartate or taurine
(references a and b). aBertran et al. (1992b); bWells and Hediger (1992); cTate et al. (1992); dBertran
et al. (1993); eLee et al. (1993).
leucine and L-methionine and sodium-independent uptake of cationic amino acids, but the
latter activity is increased by only 2-to 10-fold (Km for L-arginine near 40 mol l 1). This
activity is reminiscent of the y+L system recently described in human erythrocytes, which
exhibits sodium-independent high-affinity transport of cationic amino acids and high-
affinity transport of L-leucine in the presence of sodium (Devés et al. 1992).
The proteins rBAT and 4F2hc are less hydrophobic than common transporter
proteins
Biochemical and immunocytochemical studies have demonstrated that the rBAT and
4F2hc proteins are integral plasma membrane glycoproteins expressed in the cell
Proteins involved in amino acid transport
129
25
)
20
−
1
rBAT
15
oocyte−1
10
-Cystine uptake
L
(pmol 5 min
5
Water
0
0
50
100
150
L-Cystine ( mol l−1)
Fig. 3. Transport of L-cystine elicited by human rBAT in Xenopus oocytes. Oocytes were
injected with 50 nl of water alone (open squares) or water containing 5 ng of synthetic RNA
from human rBAT (filled squares). Three days later, the uptake of L-[35S]cystine, at the
indicated concentrations, was determined for 5 min incubations. Notice the saturation of the
rBAT-induced uptake at concentrations of L-cystine above 50 mol l 1; in contrast, uptake of
L-cystine in water-injected oocytes shows no detectable saturability at concentrations below
150 mmol l 1. Values are mean ± S.E.M. of the uptake values from seven oocytes in a
representative experiment. This experiment was performed by J. Chillarón in our laboratory.
membrane. The experimental proof for rBAT is as follows. (i) Addition of microsomes to
the reticulocyte translation system for synthetic rBAT RNA increases the mass of the
major rBAT protein product, which is sensitive to endoglycosidase H treatment
(Markovich et al. 1993; Wells and Hediger, 1992). (ii) Xenopus oocyte translation of
synthetic human rBAT RNA results in an rBAT-specific protein band ([35S]methionine
labeling) of approximately 94 kDa in crude oocyte membranes, prepared after stripping
with sodium carbonate; treatment of the oocytes with tunicamycin shifts the rBAT
translation product to a molecular mass of approximately 72 kDa (Bertran et al. 1993).
The size of this protein product is reasonably similar to the size of the protein deduced
from the predicted open reading frame (Mr approximately 79 103). A similar size for the
glycosylated protein (approximately 90 kDa) has been reported for rBAT in rat renal
brush-border membranes with anti-rBAT polyclonal antibodies (Furriols et al. 1993;
Mosckovitz et al. 1993). (iii) Immunoelectron microscopy studies show that rBAT is
expressed in the microvilli of the proximal straight tubule of the rat nephron (Furriols
et al. 1993; Pickel et al. 1993).
The main argument against the hypothesis that rBAT and 4F2hc are the actual amino
acid transporters, bo,+-like and y+L-like respectively, relies on the predicted structure of
these two proteins. The hydrophobicity plot for the human rBAT and 4F2hc proteins is
shown in Fig. 1. Only a single membrane-spanning domain is predicted around amino
acid residue 100 in the two proteins. At present, the most accepted structural model
130
M. PALACI´N
indicates that the rBAT and 4F2hc proteins are type II membrane glycoproteins (see
Fig. 4). Alternatively, Udenfriend’s group (Tate et al. 1992) have suggested that rBAT
could be arranged in the membrane with at least four transmembrane domains, with three
amphipathic -helices (see Fig. 5). Experimental evidence is needed to elucidate the
topology of rBAT insertion into cell membranes.
It is more likely that rBAT and 4F2hc proteins are modulators or components of amino
acid transport systems (Bertran et al. 1992a; Wells et al. 1992): rBAT could ‘activate’ a
silent system bo,+-like endogenous transporter of the oocyte, while 4F2hc may ‘activate’ a
y+L-like endogenous system partially ‘inactive’ in the oocyte. rBAT and 4F2hc could act
as modulators of these amino acid transporters. In this sense, recent findings suggest that
several integral membrane proteins with a single predicted transmembrane domain may
specifically modulate the action of particular channels or transporters in oocytes or
transfected cells (e.g. IsK and phospholemman, or the putative modulators of the
Na+/glucose cotransporter and intestinal peptide transport) (Attali et al. 1993; Dantzig
et al. 1994; Veyhl et al. 1993). Alternatively, rBAT and 4F2hc proteins could be essential
subunits of heteroligomeric transporters and could be associated with silent endogenous
catalytic subunits of the oocyte transporters (see Fig. 2B). The 4F2hc cell surface antigen
is a disulfide-linked heterodimer (approximately 125 kDa) composed of a glycosylated
heavy chain of 85 kDa (i.e. 4F2hc) covalently linked by disulfide bridges to a non-
glycosylated light chain of 40 kDa (Fig. 2B) (Haynes et al. 1981; Hemler and Strominger,
1982). To our knowledge, the light chain has not been cloned or microsequenced.
Similarly, rBAT seems to be linked by disulfide bridges to an unidentified putative ‘light
subunit’ within a complex of approximately 125 kDa in rat renal brush-border
membranes (C. Mora, J. Chillaro´n and M. Palacín, in preparation). In addition, expression
of rBAT in COS cells results neither in amino acid transport expression nor in the
association of rBAT into a higher molecular mass complex under non-reducing
conditions (C. Mora, J. Chillaro´n and M. Palacín, in preparation). These findings suggest
that proper functional expression of rBAT might depend on the expression of this putative
essential subunit. This hypothetical mechanism is analogous to the role proposed for the
Na+/K+-ATPase
subunit (another type II membrane glycoprotein) which, upon
injection of its RNA into oocytes, supports the maturation of active pumps containing the
endogenous catalytic
subunit (Geering et al. 1989). If this hypothesis holds true for
rBAT and 4F2hc, the structure of the mature bo,+-like and y+L-like transporters would
then be heterodimeric, a feature not yet described for transporters of organic substrates in
mammals. Elucidation of the mechanisms involved in 4F2hc and rBAT expression of
amino acid transport will require the isolation and/or cloning of the light chain of the 4F2
cell surface antigen and the putative complementary subunit of rBAT.
Identification of rBAT as a cystinuria gene
Cystinuria is an autosomal recessive disease, with an overall prevalence of one case in
7000 people, which is characterized by urinary hyperexcretion of cystine and cationic
amino acids (Levy, 1973; McKusick, 1990; Segal and Thier, 1989). Cystine has a low
solubility and its precipitation results in the formation of calculi in the urinary tract, which
Proteins involved in amino acid transport
131
leads to obstruction, infections and ultimately to renal insufficiency (Segal and Thier,
1989). Three types of classic cystinuria have been described (Rosenberg et al. 1966a):
type I, in which heterozygotes show normal aminoaciduria, and types II and III, in which
heterozygotes show cystine–lysinuria. In contrast to types I and II, type III homozygotes
show an increase in cystine plasma levels after oral cystine administration. These
different types are thought to be due to allelism of the same gene (Rosenberg et al.
1966b).
Cystinuria has been postulated to result from a defect in the high-affinity transport of
cystine, shared with cationic amino acids, through epithelial cells of the renal tubule and
intestinal tract (Rosenberg et al. 1965). Reabsorption of L-cystine in the kidney is not
completely understood. Transport of L-cystine in brush-border membrane vesicles is
relatively sodium-independent (Foreman et al. 1980; McNamara et al. 1981, 1992). The
driving force for reabsorption of L-cystine is provided by intracellular reduction to
L-cysteine, which then leaves the cell by a basolateral transport system (Silbernagl, 1988).
Studies with brush-border membrane vesicles have suggested the existence in renal
membranes of a high-affinity system, shared with cationic amino acids, and a low-affinity
system for L-cystine, which appears to be unshared (McNamara et al. 1981; Segal et al.
1977). Transport in jejunal vesicles involves a high-affinity system (Ozegovic et al.
1982), which is defective in biopsies of intestinal mucosa from cystinuric patients
(Coicadan et al. 1980; Thier et al. 1964). Functional studies indicate that the high-affinity
(micromolar range) transport of L-cystine is located in the proximal straight tubule
(Schafer and Watkins, 1984). Moreover, the high-affinity transport of L-cystine appears
to be shared with some L-zwitterionic amino acids (Foreman et al. 1980; Furlong and
Posen, 1990; Schafer and Watkins, 1984).
rBAT messenger RNA is localized within kidney and intestinal mucosa (Bertran et al.
1992b, 1993; Lee et al. 1993; Wells and Hediger, 1992; Yan et al. 1992). In keeping with
this distribution, hybrid-depletion experiments of renal and intestinal mRNA with rBAT
anti-sense oligonucleotides block expression of system bo,+-like activity in oocytes
(Bertran et al. 1993; Magagnin et al. 1992; Wells and Hediger, 1992). Fig. 4 shows two
rBAT
trancripts in kidney and small intestine, which represent alternative
polyadenylation of the same gene (Bertran et al. 1992b; Markovich et al. 1993). In situ
hybridization and immunodetection studies demonstrate specific rBAT expression in the
microvilli of small intestine and the proximal straight tubule of the rat nephron (Furriols
et al. 1993; Kanai et al. 1992; Pickel et al. 1993). Brain tissues show a longer rBAT
transcript (Fig. 4), which is also present in other human tissues (Bertran et al. 1993; Yan
et al. 1992). RNAase protection assays and immunological studies suggest that this long
transcript may correspond to a homologous neural-tissue-specific mRNA transcribed
from a different gene (Pickel et al. 1993; Yan et al. 1992).
The specific expression of rBAT in kidney and small intestine and the characteristics of
the high-affinity uptake of L-cystine induced by rBAT in oocytes (see above) suggested
that rBAT would be a good candidate for the cystinuria gene. To test this hypothesis, the
search for cystinuria-specific mutations in rBAT was undertaken (Calonge et al. 1994),
taking advantage of illegitimate transcription in lymphoblastoid cells (Chelly et al. 1989).
Amplified rBAT cDNAs from lymphoblastoid cell lines from several patients were
132
M. PALACI´N
ey
rain
Kidn
Intestine Liver
B
5.2 kb
4.1 kb
2.4 kb
Fig. 4. Tissue distribution of rBAT mRNA. At high stringency conditions, rabbit rBAT cDNA
hybridized to transcripts of approximately 2.4 kb and approximately 4.1 kb present in rabbit
kidney (i.e. cortex and medulla) and small intestine (i.e. jejunum). rBAT cDNA also
hybridized to a rabbit brain transcript of approximately 5.2 kb. rBAT-specific signals are not
visible in RNA from rabbit liver. This Northern blot was performed by M. Furriols following
the method described in Furriols et al. (1993).
analysed by single-strand conformation polymorphism, followed by direct sequencing of
electrophoretically altered fragments: six cystinuria-specific point missense mutations in
the rBAT gene, confirmed in genomic DNA, were found in 30 % of the independent
cystinuric chromosomes analyzed (Calonge et al. 1994). All these mutations affect well-
conserved amino acid residues in the human, rat and rabbit rBAT proteins. The
localization of these amino acid substitutions in the protein rBAT is indicated in Figs 2A
and 5. The main mutation found (present in seven families of cystinuric patients), M467T
(i.e. substitution of methionine at residue 467 by threonine), was detected in homozygosis
in two cystinuric kindreds (a Spanish family, Calonge et al. 1994, and an Italian family, P.
Gasparini, personal communication) and seems to be associated with type I cystinuria.
Interestingly, mutation M467T greatly impaired (approximately 80 %) L-cystine,
Proteins involved in amino acid transport
133
214
261
513
R181Q
332
575
495
Outer membrane
112
393
481
592
M467T
88
416
461
612
Inner membrane
P615T
T652R
L678P
N
C
(685)
Fig. 5. Schematic location of cystinuria-specific mutations of rBAT in the model of rBAT
containing four membrane-spanning domains. The mutations found in the rBAT gene of
cystinuric patients are described in the legend to Fig. 2A. Mutation M467T (also M467K) is
located in the third transmembrane domain proposed by Tate et al. (1992). Three mutations
(P615T, T652R and L678P) are grouped towards the C terminus. All six rBAT mutations
involve conserved or well-conserved amino acid residues in human, rat and rabbit rBAT.
Numbers indicate the first and last amino acid residues in the putative transmembrane domains
and the positions of potential N-glycosylation sites (Y).
L-arginine and L-leucine transport activity associated with rBAT in oocytes (Calonge et
al. 1994). These data provide convincing evidence that rBAT is a cystinuria gene.
The rBAT gene has been localized to the short arm of human chromosome 2 (i.e. region
2pter–p12) (Calonge et al. 1994; Lee et al. 1993). The work of Calonge et al. (1994) has
received independent and simultaneous confirmation by linkage studies with
chromosome 2p markers. Pras et al. (1994) have found linkage (maximal lod score
greater than 9) between cystinuria and microsatellite D2S119 with an approximate
location of the cystinuria locus at 7 centimorgans telomeric to this marker.
The involvement of rBAT gene in cystinuria has the following consequences. (i) As
predicted by functional and immunolocalization studies, rBAT is related to high-affinity
reabsorption of cystine in kidney. (ii) System bo,+, which transports zwitterionic amino
acids in addition to cystine and cationic amino acids, is defective in cystinuria. Why is
urinary excretion of zwitterionic amino acids not increased in cystinuria? In keeping with
the involvement of zwitterionic amino acids in cystinuria, administration of cycloleucine
to humans or rats produces increased urinary excretion of cystine and cationic amino
acids in amounts similar to those in cystinuria patients (Brown, 1967). This finding is
consistent with the inhibition caused by cycloleucine on the human rBAT-induced
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M. PALACI´N
cystine transport in oocytes (J. Chillaron, unpublished results). However, the normal
urinary excretion of zwitterionic amino acids in cystinuric patients could be explained by
the activity of intact zwitterionic amino acid reabsorption systems in the renal tubule (e.g.
system NBB).
Open questions and further perspectives
The proteins rBAT and 4F2hc introduce intriguing questions regarding amino acid
transport. (1) How do these two proteins, which apparently do not act as pore-forming
proteins, participate in specific amino acid transport activity? This question may be
answered through the elucidation of the structure of rBAT and 4F2 proteins. First, the
cloning and structural identification of the light subunit of 4F2 antigen and of the putative
‘light subunit’ of rBAT are required. Second, the topology of rBAT and 4F2hc should be
determined experimentally. (2) Is rBAT the only gene involved in cystinuria? The
hypothesis that rBAT acts as a component of the renal bo,+-like transporter suggests that
other genes might also be involved in cystinuria. Recently, one case of a de novo balanced
translocation (14;20) has been associated with cystinuria and mental retardation,
suggesting that one of these breakpoints (14q22 or 20p13) might be involved in cystinuria
(Sharland et al. 1992). In contrast, the linkage studies with chromosome 2p markers by
Pras et al. (1994) suggest the chromosome 2 locus (i.e. rBAT gene) as the only genetic
locus for cystinuria. A wider selection of cystinuria families for linkage studies, with
attention to their cystinuria phenotypes (i.e. types I, II and III), will be needed to
determine whether other genetic loci are involved in cystinuria. (3) What characteristics
do the amino acid transport activities associated with rBAT and 4F2hc show in
mammalian cells? At present, these have been investigated only in Xenopus oocytes. This
question will most probably be answered following the study of cell models that naturally
express rBAT (e.g. OK cells) and the 4F2 antigen (e.g. stimulated lymphocytes).
(4) Finally, are there any genes homologous to rBAT and 4F2hc to be identified? The
related rBAT transcript present in neural tissues may foster studies directed to the cloning
of a homologous rBAT gene in those tissues. In addition, homologous clones to rBAT and
4F2hc might be responsible for amino acid transport activities similar to those associated
with these proteins (e.g. the blastocyst system bo,+ and the sodium-dependent system
Bo,+).
I am indebted to all colleagues and PhD students who participated in the work reported
in the References (Bertran et al. 1992a,b,c, 1993, 1994; Calonge et al. 1994; Furriols
et al. 1993; Magagnin et al. 1992; Markovich et al. 1993), which form part of the basis of
the present review. I thank Dr Joan Bertran for the use of material from his PhD thesis. I
also thank Drs Antonio Zorzano and Xavier Testar and Ms M. Julia Calonge, Ms Conchi
Mora and Mr Josep Chillaron for critical reading of the manuscript. The editorial work by
Mr Robin Rycroft is acknowledged. Work performed in the author’s laboratory was
supported by grant PB90/0435 from the Dirección General de Investigación Científica y
Técnica (Spain).
Proteins involved in amino acid transport
135
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