The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Abstract

Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu218 in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based upon the published structure of the E. coli glycerol-3-phosphate transporter. No evidence was obtained for Arg306 (TM 8) of MCT1 and Glu218 of basigin forming a charge-pair; indeed E218Q-basigin could replace WT-basigin, although E218R-basigin was inactive. No PM expression of R306E-MCT1 or D302R-MCT1 was observed but D302R/R306D-MCT1 reached the PM, as did R306K-MCT1. However, both were catalytically inactive suggesting that Arg306 and Asp302 form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg86 to Glu or Gln within TM3 of MCT1 had no effect on plasma membrane expression or activity of MCT1. However, unlike WT-MCT1, these mutants enabled expression of E218R-basigin at the plasma membrane of COS cells. We propose that TM3 of MCT1 lies alongside the TM of basigin with Arg86 adjacent to Glu218 of basigin. Only when both these residues are positively charged (E218R-basigin with WT-MCT1) is this interaction prevented; all other residue pairings at these positions may be accommodated by charge-pairing or stabilization of unionized residues through hydrogen bonding or local distortion of the helical structure.

Figure 1

Homology model of rat MCT1 based on the E. coli glycerol 3-phosphate transporter (1PW4) template. The ribbon is coloured from red to purple along the sequence according to the horizontal bar (N to C). The numbered residues refer to those discussed in the text. Acidic residues (Asp, Glu) are represented by red spheres at the C-alpha position, basic residues (Arg, Lys) by blue spheres, histidine by pink spheres and aromatic residues frequently found at the phospholipid interface (Tyr, Trp) in yellow. Phe₃₀₆, that is known to be in the substrate-binding pocket, is shown in brown. The black vertical bar measures 30 Å and marks the ‘best guess’ position of the lipid bilayer. The sequence alignment of rat MCT1 with the E. coli glycerol 3-phosphate transporter used to generate the model is shown beneath the model. Lower case letters refer to residues not built in the model and residues not present in the 1PW4 crystal structure respectively. Sequence in red refers to TM-helices in the template and predicted by TMHMM in the target sequence.

Figure 2

The E218Q mutant of basigin, but not the E218R mutant, supports lactate transport by MCT1 in Xenopus oocytes. In panel A, oocytes were injected with water (0), or cRNA for MCT1 (1) in the absence or presence (A) of antisense cRNA against Xenopus basigin and cRNA for rat basigin (B). Western blots are shown for the crude plasma membrane fraction using both MCT1 and basigin antibodies. In panel B rates of L-lactate (30 mM) transport into oocytes measured using BCECF fluorescence are shown as means±SEM of 5–8 separate oocytes. Where indicated, antisense (AS) against Xenopus basigin as well as the cRNA for WT-, E218Q- or E218R-basigin was co-injected with the MCT1 cRNA.

Figure 3

The E218Q mutant of basigin, but not the E218R mutant, is correctly targeted to the plasma membrane of COS cells. COS cells were co-transfected with MCT1-c-CFP and basigin-c-YFP constructs containing the mutations indicated and live cell imaging performed as described under ‘Methods’. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 4

R306E-MCT1 and E218R-basigin are not expressed at the plasma membrane of Xenopus oocytes. Oocytes were micro-injected with the cRNA shown and after 72 hours some oocytes were used for immunofluorescence microscopy with the antibody shown (panel A) and others used for membrane preparation followed by SDS-PAGE (20 mg protein) and western blotting with anti-rat MCT1 antibody (panel B). For the western blot, kidney plasma membranes were used as a positive control. The faint band in the water-injected controls represents very slight sample contamination and is only visible because of the over-exposure of the blot to ensure any expressed MCT was detected. Further details are given under ‘Methods’. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 5

D302R/R306E-MCT1 is expressed at the plasma membrane of Xenopus oocytes but is inactive. Details are as given for Figure 4. Transport measurements are not shown because D302R/R306E-MCT1 failed to elicit any lactate transport whether or not WT- or E218R-basigin cRNA was co-injected. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 6

R306K-MCT1 is expressed at the plasma membrane of Xenopus oocytes but is inactive. Details are as given for Figure 4. Transport measurements are not shown because R306K-MCT1 failed to elicit any lactate transport whether or not WT- or E218R-basigin cRNA was co-injected. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 7

Mutation of Arg₈₆ or Arg₁₉₆ to glutamine or glutamate does not prevent MCT1 from being correctly targeted to the plasma membrane of COS cells. COS cells were co-transfected with MCT1-c-CFP and basigin-c-YFP constructs containing the mutations indicated and live cell imaging performed as described under ‘Methods’. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 8

FRET measurements suggest that mutation of Arg₈₆ perturbs the interaction of MCT1 with basigin. COS cells were cotransfected with MCT1-c-CFP and basigin-c-YFP constructs containing the mutations indicated and live cell imaging with determination of FRET performed as described under ‘Methods’. Data are presented as means±SEM for the number of observations shown.