The monocarboxylate (lactate) transporters MCT1 and MCT4 require the membrane-spanning glycoprotein CD147 for their correct plasma membrane expression and function. We have successfully expressed CD147 and MCT1 tagged on their C or N termini with either the cyan (CFP) or yellow (YFP) variants of green fluorescent protein. The tagged proteins were correctly targeted to the plasma membrane of COS-7 cells and were functionally active. Measurements of fluorescence resonance energy transfer (FRET) between all combinations of the tagged proteins were made. FRET was observed when either the C or N terminus of MCT1 (intracellular) is tagged with CFP or YFP and co-expressed with CD147 tagged with YFP or CFP on the C terminus (intracellular) but not the N terminus (extracellular). FRET was also observed between two CD147 molecules when both YFP and CFP were on the C terminus but not when both were on the N terminus or one on either end. No FRET was observed between MCT1-YFP and MCT-CFP in any combination. A wide range of controls including photobleaching were employed to confirm that where FRET was observed, it was not an artifact of direct excitation of YFP by the CFP excitation laser. It was also shown that nonspecific overcrowding of proteins did not induce FRET. Because FRET only occurs between two fluorophores if they are less than 100 Å apart and in a suitable orientation, our data provide important information on the topology of CD147 and MCT1 within the plasma membrane. The minimum configuration consistent with the data is a dimer of CD147 associating with two MCT1 molecules such that the C terminus of CD147 in the cytosol is close to the C terminus of its partner CD147 and to the C and N termini of an associated MCT1 molecule. FRET may provide a non-invasive technique for measuring changes in these interactions in living cells. The monocarboxylate (lactate) transporters MCT1 and MCT4 require the membrane-spanning glycoprotein CD147 for their correct plasma membrane expression and function. We have successfully expressed CD147 and MCT1 tagged on their C or N termini with either the cyan (CFP) or yellow (YFP) variants of green fluorescent protein. The tagged proteins were correctly targeted to the plasma membrane of COS-7 cells and were functionally active. Measurements of fluorescence resonance energy transfer (FRET) between all combinations of the tagged proteins were made. FRET was observed when either the C or N terminus of MCT1 (intracellular) is tagged with CFP or YFP and co-expressed with CD147 tagged with YFP or CFP on the C terminus (intracellular) but not the N terminus (extracellular). FRET was also observed between two CD147 molecules when both YFP and CFP were on the C terminus but not when both were on the N terminus or one on either end. No FRET was observed between MCT1-YFP and MCT-CFP in any combination. A wide range of controls including photobleaching were employed to confirm that where FRET was observed, it was not an artifact of direct excitation of YFP by the CFP excitation laser. It was also shown that nonspecific overcrowding of proteins did not induce FRET. Because FRET only occurs between two fluorophores if they are less than 100 Å apart and in a suitable orientation, our data provide important information on the topology of CD147 and MCT1 within the plasma membrane. The minimum configuration consistent with the data is a dimer of CD147 associating with two MCT1 molecules such that the C terminus of CD147 in the cytosol is close to the C terminus of its partner CD147 and to the C and N termini of an associated MCT1 molecule. FRET may provide a non-invasive technique for measuring changes in these interactions in living cells. monocarboxylate transporter 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein cyan fluorescent protein fluorescence resonance energy transfer green fluorescent protein yellow fluorescent protein polymerase chain reaction The transport of lactic acid across the plasma membrane is of critical importance to all cells. Thus for glycolytic cells such as white muscle fibers, glia cells, most tumor cells, and blood cells, as well as all other cells under hypoxic conditions, two lactic acids must exit the cell for each glucose utilized. In other tissues, lactic acid is transported into the cell for gluconeogenesis and lipogenesis (liver, kidney) or oxidation as a major respiratory fuel (heart, skeletal muscle, and brain) (1Poole R.C. Halestrap A.P. Am. J. Physiol. 1993; 264: C761-C782Crossref PubMed Google Scholar). Transport is mediated by a family of proton-linked MonoCarboxylateTransporters (MCTs)1 of which nine members have been identified (2Halestrap A.P. Price N.T. Biochem. J. 1999; 343: 281-299Crossref PubMed Scopus (1068) Google Scholar), although only for MCTs 1–4 has transport of lactic acid been demonstrated directly (3Bröer S. Schneider H.P. Bröer A. Rahman B. Hamprecht B. Deitmer J.W. Biochem. J. 1998; 333: 167-174Crossref PubMed Scopus (275) Google Scholar, 4Bröer S. Bröer A. Schneider H.-P. Stegen C. Halestrap A.P. Deitmer J.W. Biochem. J. 1999; 341: 529-535Crossref PubMed Scopus (250) Google Scholar, 5Manning Fox J.E. Meredith D. Halestrap A.P. J. Physiol. (Lond.). 2000; 529: 285-293Crossref Scopus (271) Google Scholar). Recently a new member of the MCT family has been identified with only 20–30% identity to MCT1. This acts as a sodium-independent aromatic amino acid transporter (6Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), suggesting that MCT family members may have a wider role. In the red blood cell, 4,4′-diisothiocyanostilbene-2,2′-disulfonate was found to cross-link MCT1 to GP70 (Embigen) (7Poole R.C. Halestrap A.P. Biochem. J. 1992; 283: 855-862Crossref PubMed Scopus (46) Google Scholar, 8Poole R.C. Halestrap A.P. J. Biol. Chem. 1997; 272: 14624-14628Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), a cell surface glycoprotein that is a member of a small family of closely related proteins (9Miyauchi T. Jima F. Igakura T. Yu S. Ozawa M. Muramatsu T. J. Biochem. (Tokyo). 1995; 118: 717-724Crossref PubMed Scopus (26) Google Scholar), including CD147 (also known as OX-47, EMMPRIN, HT7, and basigin) and SDR1 (10Shirozu M. Tada H. Tashiro K. Nakamura T. Lopez N.D. Nazarea M. Hamada T. Sato T. Nakano T. Honjo T. Genomics. 1996; 37: 273-280Crossref PubMed Scopus (72) Google Scholar). These are predicted to have common structural features including immunoglobulin-like domains in the extracellular region, a short C-terminal cytoplasmic tail, and a single transmembrane domain (11Fossum S. Mallett S. Barclay A.N. Eur. J. Immunol. 1991; 21: 671-679Crossref PubMed Scopus (145) Google Scholar). Although GP70 is not widely distributed, CD147 is (12Seulberger H. Unger C.M. Risau W. Neuroscience Lett. 1992; 140: 93-97Crossref PubMed Scopus (88) Google Scholar, 13Biswas C. Zhang Y. DeCastro R. Guo H. Nakamura T. Kataoka H. Nabeshima K. Cancer Res. 1995; 55: 434-439PubMed Google Scholar, 14Paterson D.J. Jefferies W.A. Green J.R. Brandon M.R. Corthesy P. Puklavec M. Williams A.F. Mol. Immunol. 1987; 24: 1281-1290Crossref PubMed Scopus (396) Google Scholar, 15Kasinrerk W. Fiebiger E. Stefanova I. Baumruker T. Knapp W. Stockinger H. J. Immunol. 1992; 149: 847-854PubMed Google Scholar). This led us to consider whether GP70 and CD147 might be required for the proper expression of MCTs. Consistent with this, we have shown that antibodies against CD-147 co-immunoprecipitated MCT1 and MCT4 but not MCT2. The interaction between CD147 and MCT1 or MCT4 was confirmed in cultured cell lines by using cross-linking antibodies against CD147 to redistribute the CD147 to one end of the cell as a cap. MCT1 and MCT4 were found to redistribute into the caps with CD147, whereas GLUT1 and MCT2 did not (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). In addition we have demonstrated that in all tissues expressing MCT1 or MCT4, CD147 is always expressed in exactly the same location (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). These observations all imply a close association between CD147 and MCT1/MCT4 within the plasma membrane, but our data also suggested that this interaction is critical for the correct expression of MCT1 and MCT4 at the cell surface. Thus when CD147 was co-expressed with MCT1 or MCT4 in mammalian cell lines, both proteins were correctly targeted to the plasma membrane, and lactate transport activity was greatly increased. In contrast, when MCT1 or MCT4 was expressed in the absence of CD147, the proteins accumulated in the endoplasmic reticulum/Golgi apparatus rather than being expressed in active form in the plasma membrane (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). Furthermore, we have shown that co-injection of Xenopus oocytes with MCT1 cRNA and an antisense cRNA to a Xenopus homologue of CD147 reduced MCT1 expression and activity (17Meredith D. Halestrap A.P. J. Physiol. (Lond.). 2000; 526P: 23PGoogle Scholar). These data imply that CD147 acts as a chaperone for MCT1 and MCT4 translocation to the plasma membrane. To study such interactions in a living cell in real time a non-invasive technique is required. In this paper we describe how this can be using fluorescence resonance energy transfer (FRET) between the two proteins tagged on their C or N termini with either the cyan (CFP) or yellow (YFP) variants of FRET between two fluorophores only occurs if they are less than 100 Å apart and in the it is to provide information interactions and associated changes that may R. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). FRET has been to study interactions between proteins or a membrane protein and a protein partner (6Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 2001; 19: PubMed Scopus Google Scholar, K. R. B. 1998; PubMed Scopus Google Scholar, A. M. F. R. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. K. Y. T. A. M. 2001; PubMed Scopus Google Scholar), but have successfully employed FRET to study the interaction of membrane proteins in their C. J. T. Biol. 2001; PubMed Scopus Google Scholar, C. A. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, P. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). To our this is the the of FRET to an interaction between two proteins that functionally in the plasma membrane, although of the transporter and the acid transporter has been in this P. M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). We both MCT1 and CD147 in CFP and YFP expression that expression of the or and a of in The for these is in In or the and and and the C-terminal tagged and the CD147 and the of into the mammalian expression (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar), was by using by the addition of an at the end and of the addition of a at the end. were with and in the correct into the of the and A was for and but the and were both and MCT1 was by the and in into the and and were by with that at the and were with and and in the correct into the of the and The were by and expressed in cells by a as cells were in with 100 and 100 FRET were in and with cells well in of whereas for of lactate were with cells in of were at in a they were and was as (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). was with of in 100 of and for being to the cells and for this the was and was an were at in using one of two The of was with a to a and with an and to photobleaching a was employed with excitation for and was using In both a was of FRET was by of the of of the fluorescence at to when at the CFP excitation The that may this and the of are in under of transport into and cells were at the of using the fluorescent 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein as (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). was at and was at and was as an in the fluorescence The of this fluorescence is directly to the of transport expressed in of and was by the data to a as Halestrap A.P. Biochem. J. PubMed Scopus Google Scholar). Xenopus with was by of the cRNA by in and of lactate transport were at using fluorescence as Fox J.E. Meredith D. Halestrap A.P. J. Physiol. (Lond.). 2000; 529: 285-293Crossref Scopus (271) Google Scholar). To confirm that of a to MCT1 not the of the to as a lactate we expressed MCT1 with a C-terminal in Xenopus Transport activity was the of in that proton-linked lactate using as under are shown in and confirm that the is lactate in a to MCT1 but with a reduced Thus the of was with a of for MCT1 Fox J.E. Meredith D. Halestrap A.P. J. Physiol. (Lond.). 2000; 529: 285-293Crossref Scopus (271) Google Scholar). one of the was successfully expressed in cells and be in cells by are in when expressed these did not a membrane plasma membrane but rather to be associated with the endoplasmic This was the for MCT1 in with our observations with MCT1 (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). can be for and in In contrast, when both an MCT1 and a CD147 were both proteins a plasma membrane location as in and and D. this is the same as observed for the proteins (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google and that the CFP and YFP to the C and N termini not with the correct of the of photobleaching to confirm is were with either and or and and using either the with excitation using the and lines as in or the with excitation at and was and the YFP by to the for or the excitation for are shown for both the CFP and YFP and with of the of the to that These data are as for or cells. are for the cell whereas for only a small of the plasma membrane was as on the of COS-7 cells expressing and or in combination. with the was as under were using the using the and lines of the to CFP and In each the of the for the cell shown was the range of In the cells with and both the in and in that FRET is This was not observed when cells were with and and as shown in To confirm that lactate transport was in cells expressing both of transport were in a of cells using This was with cells or cells with either or with that such only expression of the tagged proteins in a of the in and 20–30% in cells. Thus it was important to between and cells when of transport by cells. the of the for transport was not to this directly the CFP and YFP when of transport were it was that in cells with both and greatly of transport as shown in This is consistent with these cells expressing both and in an active The of and proteins were in cells with a single and with the lines for CFP and YFP and for and are in A and and that the two are with at and the of cells expressing both CFP and YFP were at FRET a in the of the CFP be observed as direct energy transfer occurs the CFP to the YFP with of the fluorescent at rather than In cells expressing both and this was observed as a in the and an in the with that with the CFP as shown in Thus the of to acts as an of the of FRET. it is not to the of FRET this YFP is also by at a of that at the of the to be less than in the absence of and the of this be on the expression of the YFP to the CFP if the expression of the YFP protein were to be greatly in of the CFP between such and real FRET a this were to that expression of the YFP was less than or to that of the CFP This was most by using MCT1 tagged with CFP and CD147 tagged with although for all combinations were In we data for the for all combinations of CFP and YFP The in which the is with of of (6Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (202) Google for with (6Kim D.K. Kanai Y. Chairoungdua A. Matsuo H. Cha S.H. Endou H. J. Biol. Chem. 2001; 276: 17221-17228Abstract Full Text Full Text PDF PubMed Scopus (202) Google for with Fox J.E. Meredith D. Halestrap A.P. J. Physiol. (Lond.). 2000; 529: 285-293Crossref Scopus (271) Google for with and Fox J.E. Meredith D. Halestrap A.P. J. Physiol. (Lond.). 2000; 529: 285-293Crossref Scopus (271) Google for with Because the C terminus of CD147 and both the C and N terminus of MCT1 are these all in which both fluorescent are This is consistent with FRET as a of the close of the C terminus of CD147 to both the C and N termini of MCT1. Furthermore, the are of whether the CFP and YFP are on CD147 or MCT1 as be predicted for FRET. In contrast, were when was co-expressed with or when other combinations of CD147 tagged with either CFP or YFP and either or tagged MCT1 were co-expressed These are to in which FRET is not and as important This is when CD147 has its CFP or YFP to the N terminus that is extracellular and either the C or N terminus of MCT1 in the FRET is on the two fluorophores being within Å of each other R. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar), and this not be under such for all CFP and of MCT1 and were with the combinations of and MCT1 and CD147 and the for at and was for excitation with the In such as with or with MCT1 or CD147 was also to correct plasma membrane of the tagged cells were only when correct of both tagged proteins to the plasma membrane was observed, this was also a that both proteins were expressed at Full were with excitation at and the for at and was are expressed as of the of observations and the of the fluorophores is to of the FRET is by in a new were with the combinations of and MCT1 and CD147 and the for at and was for excitation with the In such as with or with MCT1 or CD147 was also to correct plasma membrane of the tagged cells were only when correct of both tagged proteins to the plasma membrane was observed, this was also a that both proteins were expressed at Full were with excitation at and the for at and was are expressed as of the of observations and the of the fluorophores is to of the FRET is by To be that the for FRET is we have employed photobleaching of the YFP using a to the YFP excitation CFP and YFP are photobleaching of the YFP to an in the of the CFP protein. In we that this is the fluorescence were for these In a fluorescence was in which of the excitation be for the CFP and YFP excitation using of and The of the CFP excitation YFP this was not with the that was by the It can be that of the the of the increased. were for the FRET it be that the in to the were when the were with the In this the fluorescence a small of the plasma membrane was to that only targeted proteins were We have also whether FRET occurs between and or between and when these proteins are expressed this it was also to CD147 or MCT1 with the two tagged MCT1 or CD147 to correct translocation of all proteins to the plasma membrane. are shown for all combinations in as fluorescence These data imply that FRET occurs between and but not between either and and or and MCT1 was for FRET between any of CFP and YFP It be that the FRET was as a of the close of proteins that are as a of Although this is in of the controls the was by CFP and YFP of and MCT1. is single member of the that we have shown is to for CD147 as a chaperone for correct MCT1 expression (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar). We found that the most suitable of expression were using the of with and data for these are shown in It is that is for FRET under these conditions, both proteins being well It is that transporters require proteins for their expression and correct function. Thus CD147 is associated with the lactate transporters MCT1 and MCT4 and is required for their correct expression on the cell surface (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar), whereas the chain with and expression of a family of amino acid transporters B. R. J. F. 1998; PubMed Scopus Google Scholar, Y. H. K. H. E. Endou H. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, R. B. C. F. EMBO J. 1999; PubMed Scopus Google Scholar, D. J. M. R. R. P. A. K. A. P. M. 1999; 21: PubMed Scopus Google Scholar). the translocation of the to the plasma membrane of Xenopus oocytes is greatly by of which may also the activity of Y. B. PubMed Google Scholar). Furthermore, the glycoprotein and a protein have been in acid transport across the plasma membrane C. A. J. Res. 1998; Full Text Full Text PDF PubMed Google Scholar). and have also been to with proteins that can their H. A. 1999; PubMed Scopus Google Scholar, A. Brown J. R. 1998; PubMed Scopus Google Scholar). To study these interactions in living cells a non-invasive technique is and in this paper we describe how this can be using FRET between CFP and YFP of MCT1 and data that FRET is observed when either the C or N terminus of MCT1 is tagged with CFP and YFP and co-expressed with CD147 tagged with YFP or CFP on the C terminus but not the N We have a wide range of controls to confirm the of the FRET data and to the that our data are an artifact of of the CFP excitation to direct excitation of These controls the of which of expression and also YFP photobleaching that an of the CFP The that FRET was the of nonspecific interactions between proteins by overcrowding was by the of to FRET with Furthermore, FRET was observed when and were co-expressed with CD147 to that both the tagged proteins were targeted to the plasma membrane In the absence of CD147 both the CFP and accumulated in the but was of FRET not In the of these controls we that CD147 and MCT1 in such a that CFP or YFP on either the C or N terminus of MCT1 can FRET with a fluorescent partner on the C terminus of CD147 but not its N This that the C terminus of CD147 is within Å of the C and N termini of both of which are also In contrast, as be the N terminus of CD147, which is not in this data also us to how two MCT1 molecules or two CD147 molecules with each of CFP and YFP of CD147 that two CD147 molecules must be in close such that their C termini but not their N termini with each These data imply that CD147 is in the membrane as a dimer In such interactions are observed between any of and that their C and N termini are to be a that is consistent with our data of how MCT1 and CD147 may in the plasma membrane. The of the transmembrane only be as it is on that of the of has been 2000; PubMed Scopus Google Scholar). We that each MCT1 with a single CD147 that in a dimer with a how cross-linking antibodies against CD147 both the CD147 and MCT1 to as a single at one end of the cell (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar), which not be if the MCT1 with a single CD147 and its such as immunoglobulin-like domains in the extracellular region, a short cytoplasmic tail, and a single transmembrane domain (11Fossum S. Mallett S. Barclay A.N. Eur. J. Immunol. 1991; 21: 671-679Crossref PubMed Scopus (145) Google Scholar). The transmembrane a of W. Fiebiger E. Stefanova I. Baumruker T. Knapp W. Stockinger H. J. Immunol. 1992; 149: 847-854PubMed Google including a acid A amino acid within the transmembrane is a common of proteins with transmembrane domains M.H. PubMed Google but for a protein with a single transmembrane domain 1991; PubMed Scopus Google Scholar), an important function. we have (16Kirk P. Wilson M.C. Heddle C. Brown M.H. Barclay A.N. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google that critical for the association of the two proteins is an interaction between this transmembrane domain of CD147 and a transmembrane domain of MCT1. This might be that a known to be for transporter activity B. Schneider H.P. Bröer A. Deitmer J.W. Bröer S. 1999; PubMed Scopus Google Scholar). we have a to of and found that this the plasma membrane MCT1 to be expressed at the plasma membrane not The of that the transmembrane domain of CD147 and of MCT1 are closely This the C terminus of CD147 amino and the N or C terminus of MCT1 and to be close for FRET to between the CFP and YFP We for in the and FRET
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