(Received for publication, December 23, 1996, and in revised form, April 1, 1997)
From the Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom
Treatment of intact erythrocytes with
4,4-diisothiocyanostilbene-2,2
-disulfonate (DIDS) causes irreversible
inhibition and chemical labeling of the lactate transporter,
monocarboxylate transporter 1 (MCT1) (Poole, R. C., and Halestrap, A. P. (1992) Biochem. J. 283, 855-862). In rat eythrocytes
DIDS also causes cross-linking of MCT1 to another protein in the
membrane to give a product of 130 kDa on SDS-polyacrylamide gel
electrophoresis. Cross-linking is markedly reduced by those compounds
that protect against irreversible inhibition of lactate transport by
DIDS and enhanced by imposition of a pH gradient across the plasma
membrane to recruit the substrate binding site of MCT1 to an exofacial conformation. These data indicate that DIDS cross-linking is via the
same site on MCT1 as is responsible for inhibition of transport. Antibodies raised against the cross-linked conjugate react with proteins of approximately 40 kDa (MCT1) and 70 kDa on Western blots of
erythrocyte membranes and an additional band of 130 kDa after treatment
of erythrocytes with 100 µM DIDS. The 70-kDa protein that
is cross-linked to MCT1 was purified and shown to contain N-linked carbohydrate; the apparent core molecular mass is
40 kDa. Amino acid sequencing showed that the protein is the rat equivalent of the membrane-spanning mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin superfamily related to basigin (Ozawa, M., Huang, R. P., Furukawa, T., and Muramatsu, T. (1988) J. Biol. Chem. 263, 3059-3062). Possible implications
of the specific interaction between MCT1 and this protein are
discussed.
Proton-monocarboxylate transporters
(MCTs)1 are essential for the well being of
almost all mammalian cells and play a central role in the transport of
lactate between tissues (1). Recently two distinct MCTs (MCT1 and MCT2)
have been cloned from mammalian cells (2-6). MCT1 is widely
distributed in mammalian cells, and has been well characterized at the
functional (1, 7) and structural level (8), especially in erythrocytes
(1, 8). It is inhibited by a variety of stilbene disulfonates, such as 4,4-dibenzamidostilbene-2,2
-disulfonate (DBDS) and
4,4
-diisothiocyanostilbene-2,2
-disulfonate (DIDS), which bind in
competition with substrates and some other inhibitors of transport (9).
DIDS has two isothiocyanate groups that are reactive toward susceptible
amino groups, and with prolonged incubation these cause irreversible
inhibition and chemical labeling of the transporter (9, 10). The two
isothiocyanate groups of DIDS give it the potential to cross-link MCT1
with closely associated membrane proteins. Indeed, SDS-PAGE of rat
erythrocyte ghosts prepared from cells preincubated with DIDS revealed
a protein band of approximately 130 kDa that might be such a
cross-linked product (10). Here we show that this 130-kDa band is the
result of a highly specific DIDS-mediated cross-linking of MCT1 to a glycoprotein of approximately 70 kDa. Internal sequencing of the protein shows that is is the rat equivalent of the mouse
teratocarcinoma glycoprotein, GP-70, which is a member of the
immunoglobulin gene superfamily related to basigin (11). Cross-linking
appears to occur via a site at or in communication with the substrate
binding site of MCT1 and might play a role in regulating MCT1
activity.
Materials
Chemicals and biochemicals were obtained from the sources given previously (8, 9, 12). The protease Lys-C was from Sigma, Poole, Dorset, UK. Anti-peptide antibodies to various regions of MCT1 were raised and purified as described elsewhere (8).
Methods
Labeling of Erythrocytes with DIDS and Analysis of ProductsErythrocytes were collected in a citrate buffer (84 mM sodium citrate, 1 mM EGTA, pH 7.4). Cells were then usually washed once in a bicarbonate-buffered saline buffer (121 mM NaCl, 25 mM NaHCO3, equilibrated with 95% O2, 5% CO2), and then at least twice more in citrate buffer before resuspending in the same buffer to 10% hematocrit and adjusting the pH to 7.4. Incubations with DIDS, removal of nonbound inhibitor, and preparation of ghost membranes were performed as described previously (10). Proteolytic digestion of red cell ghosts and separation of membrane proteins by SDS-PAGE were performed as described elsewhere (8).
Anti-(MCT1-70-kDa Binding Protein Conjugate) AntibodyRat erythrocyte membranes, prepared from cells treated with 100 µM DIDS, were subjected to chromatography on aminoethyl-Sepharose, essentially as described previously (12). Elution of the cross-linked product of 130 kDa was monitored both by Western blotting with anti-MCT1 antibodies, and by silver staining of protein. The peak fractions containing the cross-linked product (which co-eluted with free MCT1) were concentrated by centrifugal filtration (Amicon, Centriprep 10) and separated on a 6% (w/v) SDS-PAGE gel, and the protein was located with copper staining (13). The band of 130 kDa was excised, destained, and then electroeluted as described below. This preparation was used to immunize a New Zealand White rabbit, as described previously (8).
Purification of the 70-kDa Binding Protein and Sequencing of Lys-C Cleavage PeptidesThe membrane protein of approximately 70 kDa that is cross-linked to MCT1 upon incubation of rat erythrocytes with DIDS was shown to co-elute with MCT1 on ion-exchange fractionation (Q-Sepharose). Thus membranes (from either control or 5 µM DIDS-treated erythrocytes) were stripped of peripheral membrane proteins and solubilized with 1% (w/v) C12E8 at a protein concentration of 1 mg/ml, prior to batch purification using Q-Sepharose (10). Nonbound protein was removed by washing with buffer containing 0.5% C12E8, and a 0.2 M NaCl eluate was prepared by mixing the washed matrix with an equal volume of buffer containing 0.5% C12E8 and 0.4 M NaCl. The eluate was concentrated by centrifugal filtration to 2 ml, and proteins were separated by SDS-PAGE. A broad band at approximately 70 kDa (identified by staining with Coomassie Blue) was electroeluted overnight in buffer containing 20 mM Tris, 2 mM EDTA, 0.1% (v/v) 2-mercaptoethanol, and 0.1% SDS (w/v), pH 8.0, before concentrating to 1.5 ml by centrifugal filtration. C12E8 was added to 1% (v/v), and the protein was incubated with N-glycanase F (400 Oxford Glycosystems units/ml) for at least 2 h, and usually overnight. The sample was then subjected to SDS-PAGE, where the deglycosylated binding protein migrated at approximately 40 kDa. Direct N-terminal microsequencing was performed after transfer onto a ProBlot membrane and staining with Serva Blue G (12). Alternatively, the polyacrylamide gel was stained with Coomassie Blue, and the 40-kDa band was excised, electroeluted overnight, and concentrated by centrifugal filtration. After addition of C12E8 (1% (v/v) in a final volume of 200 µl), a 50-µl aliquot was incubated overnight at 37 °C with 0.2 µg of Lys-C, and the products were separated by SDS-PAGE using a Tricine buffer system able to separate small peptides (14). Peptides were transferred onto ProBlot membrane and stained with Serva Blue G to reveal discrete bands of about 11 and 3.5 kDa. These were cut out for N-terminal microsequencing.
The
data of Fig. 1 show that the 130-kDa band formed
following incubation of rat erythrocytes with 100 µM DIDS
is recognized by specific anti-MCT1 antibodies. It is of note that this
cross-linking is highly specific; normally no other additional bands
were detected upon treatment with DIDS. These observations indicate
that the cross-linking is probably a reflection of a close association of the two proteins in the erythrocyte membrane, which may in turn be
of functional relevance. Since DIDS is membrane impermeant, the binding
protein must be either a membrane-spanning protein or an exofacial
peripheral protein. It could be argued that the new MCT-containing band
is a result of cross-linking MCT1 to itself, forming oligomers. This
seems unlikely for several reasons. First, 130 kDa is not a simple
multiple of 40 kDa (especially as trimer formation seems unlikely), and
second, the products of self-aggregation of MCT1 do not seem to
co-migrate with the cross-linked product as is also shown in Fig. 1.
Aggregation was induced by solubilizing rat erythrocyte membranes with
the detergent C12E8 (1% w/v) and leaving on
ice for varying periods of time before separating on SDS-PAGE and
probing Western blots with anti-MCT1 antibodies. There was a
time-dependent aggregation of MCT1 to produce a product of
approximately 80 kDa, which corresponds to a dimer of MCT1. Such
aggregation phenomena are common with membrane proteins (15, 16).
To investigate more fully the cross-linking reaction, we performed the
labeling reaction in the absence and presence of various inhibitors of
MCT1 activity. DBDS and CHC, two potent inhibitors of lactate transport
that also inhibit labeling of MCT1 by DIDS (9, 10), reduced markedly
the DIDS-induced cross-linking of MCT1, whereas the poor inhibitor
4,4-dinitrostilbene-2,2
-disulfonate (DNDS) (9) had little effect
(Fig. 2). These results demonstrate that the
cross-linking of MCT1 is via a site on the transporter that is either
at the substrate binding site, consistent with competitive inhibition
of lactate transport by DIDS, or affected by conformational changes
induced by substrate or inhibitor binding. In Fig. 3 we
show that the cross-linking reaction is dependent upon the nature of
the buffer used for the reaction. The reaction was performed in either
a citrate buffer, pH 7.4, which induces a large pH gradient (0.6-0.8
pH unit, alkaline inside) or a saline buffer, also at pH 7.4, in which
there is very little gradient (approximately 0.2 pH unit, acid inside)
(17). It is clear that in citrate buffer the rate of cross-linking of
MCT1 is more rapid and the extent is greater. This result might be
expected since an alkaline-inside pH gradient would be predicted to
cause recruitment of an outward-facing binding site of the transporter,
according to the accepted kinetic model for the carrier in which proton binding precedes monocarboxylate binding (1). Thus the empty carrier
will preferentially take up the conformation in which the substrate
binding site is exposed to the face of the membrane at which the proton
concentration is highest. The inhibitor then binds and traps the
carrier in this conformation. This model is supported by the
observation that the Ki for inhibition of lactate
transport by the reversibly binding stilbene disulfonate DBDS is lower
in citrate than in saline buffer (results not shown). Such data provide
further evidence that the cross-linking occurs at or near the external
substrate binding site.
The cross-linking reaction can also be used to locate the region of
MCT1 containing the DIDS-labeled lysine residue, by investigation of
the fragments produced upon proteolysis of MCT1. Using a series of
anti-peptide antibodies, we have characterized the pattern of cleavage
by several proteases and used this information to derive information on
the topology of the protein with respect to the membrane (8). Rat MCT1
has only 5 lysine residues predicted to be extracellular in location,
and these are the most likely targets for labeling by DIDS. Two of
these lysine residues are within a well characterized N-terminal
fragment, and the remaining three in fragments reactive with antibodies
raised against the TM7/8 and TM11/12 loops. Thus, if the binding
protein is linked to either of these fragments by DIDS, there should be
additional immunoreactve fragments derived from proteolytic digestion.
Data demonstrating this are shown in Fig. 4. Trypsin
digests MCT1 quantitatively to yield a fragment of approximately 20 kDa
that reacts with the TM11/12 antibody. Upon digestion of membranes from
DIDS-pretreated erythrocytes, additional antibody reactive bands of
approximately 100 and 35 kDa were detected. These data indicate that
DIDS reacts with a lysine in the C-terminal half of the protein, which,
following trypsin cleavage, remains conjugated to the binding protein
(100 kDa) or a proteolytically cleaved fragment of it (35 kDa).
The Binding Protein Is the Rat Homologue of the Mouse Teratocarcinoma Glycoprotein, GP-70
Membranes from DIDS-treated
erythrocytes were treated with N-glycanase F, which resulted
in an apparent reduction in molecular mass of the binding protein on
SDS-PAGE, to approximately 100 kDa (Fig. 5). Since MCT1
is not glycosylated (6), this must reflect N-linked
glycosylation of the binding protein. Antibodies raised against the
MCT1-binding protein conjugate, as described under "Experimental
Procedures," recognized two proteins on Western blots of control
erythrocyte membranes. These were a weak band at 40 kDa that reacted
with antibodies against MCT1 (not shown) and a stronger band at 70 kDa,
which is likely to represent the unconjugated binding protein (Fig.
6). In membranes prepared from cells pretreated with 100 µM DIDS, these bands were still detected, but an
additional band at 130 kDa was detected that presumably represents the
DIDS cross-linked MCT1 conjugate to which the antibody was raised.
We attempted to purify the putative 70-kDa binding protein by replacing
DIDS as cross-linker with the cleavable membrane impermeant cross-linking reagent
3,3-dithiobis[sulfosuccinimidyl]-propionate (DTSSP). Preliminary
experiments showed that erythrocytes incubated for 1 h at 37 °C
with 1 mM DTSSP gave a 130-kDa cross-linked product that
reacted with MCT1 antibody on Western blots. Partial purification of
this protein was performed on aminoethyl-Sepharose and SDS-PAGE, followed by electroelution of the 130-kDa band as described for the
DIDS-labeled conjugate under "Methods." Cleavage of the eluted protein was achieved by incubation with
-mercaptoethanol (5% v/v)
for 45 min, and the cleaved products were separated by SDS-PAGE. A
broad band of about 70 kDa was obtained, but N-terminal sequencing showed it to contain more than one polypeptide. Thus we attempted to
purify the native binding protein.
Western blotting with the antibody raised against the 130-kDa
MCT1-binding protein conjugate showed that the 70-kDa putative binding
protein eluted from Q-Sepharose at a similar salt concentration to MCT1
(0.2 M NaCl in batch elution experiments). The eluted protein fraction was further purified by SDS-PAGE, and a broad band
around 70 kDa, presumably composed of a number of polypeptides, was
electroeluted and then subjected to treatment with
N-glycanase F. The 70-kDa band, now free of MCT1, was
clearly detected with the antibody, and treatment with
N-glycanase F reduced its apparent molecular mass to 42 kDa.
Silver staining of these fractions revealed the 42-kDa band to be a
major component of the deglycosylated samples as shown in Fig.
7. The core (deglycosylated) protein prepared in this
manner was subjected to N-terminal microsequencing, but no sequence was
obtained. Thus it was digested with the protease Lys-C, and the
resulting peptides were separated by SDS-PAGE before transferring to
ProBlot membrane for N-terminal microsequencing. Two peptides were
visible of approximately 12 and 3.5 kDa, but only the 3.5-kDa peptide
gave a good sequence of 15 amino acids (MGDTLYNQYRFTVFN). This sequence
was used in a BLAST search of the protein sequence data bases via the
National Center for Biotechnology Information (NCBI) and was found to
have a strong identity to an internal sequence of mouse teratocarcinoma
glycoprotein GP-70 (SWISS-PROT accession no. P21995[GenBank]) as shown in Fig.
8. This 70-kDa glycoprotein is a cell surface member of
the immunoglobulin superfamily that has a developmentally regulated
carbohydrate moiety (11). It deglycosylates to yield a core protein
with a mobility on SDS-PAGE corresponding to about 40 kDa; the true molecular weight being 37,102. Thus it is probable that our
MCT1-binding protein is the rat equivalent of mouse GP-70, or a closely
related protein. The identity is further strengthened by the presence of a lysine on the N-terminal side of the methionine of the GP-70 peptide, since our peptide was derived by Lys-C cleavage.
Confirmation that the MCT1-binding protein is the rat equivalent of the mouse GP-70 was obtained by searching the EST data base with the GP-70 nucleotide sequence. Two closely related EST sequences were identified (EMBL accession nos. H32620[GenBank] and H32724[GenBank]) of which only the latter corresponded to the coding region of mouse GP-70. Fortunately this 276-base pair EST fragment encoded the region of the protein corresponding to the peptide we had sequenced. The translated sequence (in the third reading frame) is shown in Fig. 8 and agrees with our experimentally determined sequence in all but one residue. The incorrect residue was a Ser which we determined as an Asn. This is a common sequencing error since the two amino acids eluted with very similar retention times, but it may also be the result of a single base sequencing error in the EST sequence.
General ConclusionsThe data presented here demonstrate that DIDS can cross-link MCT1 to a 70-kDa glycoprotein in rat erythrocyte membranes via an externally disposed amino acid side chain. This protein is likely to be the rat equivalent of mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin superfamily. Since DIDS is an affinity label that binds in competition with substrates and inhibitors of MCT1, it is likely that cross-linking occurs at the substrate binding site. However, it is possible that binding occurs at a distant site whose conformation and thus affinity for DIDS is affected by substrate binding. Close apposition of GP-70 to the substrate binding site of MCT1 raises the possibility that this interaction may have an effect on the kinetic/functional properties of MCT1. In cardiac myocytes two functionally distinct monocarboxylate transporters have been detected (18-20), and neither of them have properties that correspond exactly to those of MCT1. However, there is a high level of expression of MCT1 in the intercalated disk region of the myocyte (2).2 Such apparently contradictory observations may be reconciled if the absence or presence of a regulatory protein in different cells could change the functional properties of MCT1.
Studies of the expression of GP-70 in mouse tissues have not been described in detail, although it has been reported that adult tissues express much less of the protein than embryonic tissues (11). However, GP-70 is very closely related to another membrane glycoprotein of the immunoglobulin superfamily called basigin. This protein, whose glycosylation state can vary considerable between different tissues, is also known in other species as OX-47 or CE9 (rat), HT7 or neurothelin (chick), and M6 or EMMPRIN (human) (21-23). The function of these proteins is unclear, but it has been suggested, with little evidence, that they are involved in cell signaling or adhesion. Their expression is up-regulated by stimuli that enhance the metabolic activity of cells; for example thyroid hormone in hepatocytes and cold exposure in brown fat (22). OX-47 is found in most cells, but is almost totally absent in blood cells, which suggests that GP-70 may be fulfilling an equivalent role in rat erythrocytes. In contrast, in rabbit erythrocytes, basigin is present and was first identified as a protein reactive against the "4D4" monoclonal antibody that was raised during studies on the monocarboxylate transporter by Donovan and Jennings (24). Indeed it was originally thought that this protein might be the transporter, but later studies showed that carrier activity could be separated from the 4D4 antigen (1). Thus it is possible that in rabbit erythrocytes it is basigin that fulfills the same function that GP-70 does in rat erythrocytes. However, we have not been able to detect a DIDS cross-linked MCT1-conjugate protein in rabbit erythrocytes (10), which suggests that, if basigin does interact with MCT1, lysines on the two molecules are not sufficiently close to allow cross-linking to occur.
Whether the interaction of GP-70 with MCT1 can regulate its activity remains to be established. An increased expression of OX-47 is observed in several cell types under conditions of metabolic activation that require enhanced glycolysis and are accompanied by an increase in the expression of glucose transporters (22). It is an attractive possibility that OX-47 and GP-70 may be involved in a parallel stimulation of monocarboxylate transport. There is evidence that a similar regulatory mechanism may be involved in the stimulation of neutral amino acid transport by system A in response to hypertonic shock (25). Another possibility might be that GP-70 is involved in the translocation of MCT1 from the endoplasmic reticulum to the Golgi apparatus, a role played by glycophorin in the expression of band 3 at the cell surface (26, 27). These possibilities await further investigation, by co-expression of the two proteins in cells lacking endogenous lactate transporter activity.
We thank Dr. Will Mawby for peptide sequencing.