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 (Glu 218 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 Arg 306 (TM 8) of MCT1 and Glu 218 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 Arg 306 and Asp 302 form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg 86 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 Arg 86 adjacent to Glu 218 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.

Essential Molecular Determinants for Thyroid Hormone Transport and First Structural Implications for Monocarboxylate Transporter 8

“…Moreover, this is in agreement with recent results on MCT1, where the involvement of arginine Arg 306 (TMH8, position 8.50) for transport activity was found, which corresponds to Arg 445 (TMH8, position 8.50) in MCT8. In addition, on MCT1, the negatively charged residue Asp 302 at the same TMH8 (position 8.46) was also identified to contribute in the transport mechanism (23,24,38). Guided by our MCT8 model we found on MCT8 a further charged amino acid, Asp 498 (TMH10, position 10.49), which may cooperate with Arg 445 in positioning the invariate L-amino acid moiety of the substrate.…”

“…With small deviations, the helix dimensions observed in the GlpT crystal structure are applicable for human MCT8. The reliability of this structural template is supported by examples of modeling for other members of the MFS-like GLUT1 (22) or MCT1 (23,24 Comparison with loop conformations extracted from the PDB by the SuperLooper bioinformatics software were also performed to find superimposing loop structures from the PDB as criteria for reliability. The quality and stability of the model were validated by checking the geometry using PROCHECK (25).…”

“…The MCT sequence alignment revealed that all MCTs carry a conserved arginine in TMH8 (Arg 445 , position 8.50). In MCT1, the corresponding amino acid (Arg 306 ) is described to be essential for transporter function (23). We built a homology model for MCT8 based on the x-ray structure of GlpT (Fig.…”

Overview of the Proton-coupled MCT (SLC16A) Family of Transporters: Characterization, Function and Role in the Transport of the Drug of Abuse γ-Hydroxybutyric Acid

“…The initial association of CD147 and MCT1 is required for the translocation of MCT1 to the plasma membrane (48). Furthermore, studies indicate that covalent modification of CD147 results in inhibition of lactate transport as is seen with pCMBSmediated inhibition of transport (48,50). In addition to MCT1, CD147 functions as an ancillary protein for MCT4 but not MCT2 (48).…”

Oral Absorption, Intestinal Metabolism and Human Oral Bioavailability

“…The driving force for transport is provided by both the substrate and H + -concentration gradients, with the pH gradient determining the extent of transport activity (Juel 1997;Halestrap and Price 1999). Based on the reported crystal structures of two members of the major facilitator superfamily, the Escherischia coli glycerol-3-phosphate transporter (G1pT) and lactose permease (Lac Y) (Abramson et al 2003;Huang et al 2003), the structure of MCT1 has been modelled (Manoharan et al 2006). Futhermore, site-directed mutagenesis identifying key substrate-binding residues together with structural modeling has lead to the suggestion of a translocation cycle as the mechanism of transport for MCT1 (Wilson et al 2009).…”