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

Figures & data

Supplementary Table I. Primers used for the generation of site-directed mutations in MCT1 and basigin. For site-directed mutations the underlined bases represent the change in nucleotide sequence introduced to provide the necessary mutation.

Construct	Forward primer	Reverse primer
MCT1 K38Q	5′ CCTATGCATTTCCCCAATCCATCACTGTC 3′	5′ GACAGTGATGGATTGGGGAAATGCATAGG 3′
MCT1 K45Q	5′ CCATCACTGTCTTCTTTCAAGAGATTGAAATTATAT 3′	5′ ATATAATTTCAATCTCTTGAAAGAAGACAGTGATGG3′
MCT1 (D302R +R306E)	5′ GTTAGAATGGTGGCCGAACCGTCCATGGGTCTT 3′	5′ AAGACCCATGGACGGTTCGGCCACCATTCTAAC 3′
MCT1 R86Q	5′ GAATAAATATGGCAGCCAGCCAGTAATGATTGCTGG 3′	5′ CCAGCAATCATTACTGGCTGGCTGCCATATTTATTC 3′
MCT1 R86E	5′ GAATAAATATGGCAGCGAGCCAGTAATGATTGCTGG 3′	5′ CCAGCAATCATTACTGGCTCGCTGCCATATTTATTC 3′
MCT1 R196Q	5′ TGGATCCCTGATGCAACCAATAGGGCCTC 3′	5′ GAGGCCCTATTGGTTGCATCAGGGATCCA 3′
MCT1 R196E	5′ TGGATCCCTGATGGAACCAATAGGGCCTC 3′	5′ GAGGCCCTATTGGTTCCATCAGGGATCCA 3′
MCT1 R306E	5′ GTTGATATGGTGGCCGAACCGTCCATGGGTCTT 3′	5′ AAGACCCATGGACGGTTCGGCCACCATATCAAC 3′
MCT1 R306K	5′ GTTGATATGGTGGCCAAACCGTCCATGGGTCTT 3′	5′ AAGACCCATGGACGGTTTGGCCACCATATCAAC 3′
MCT1 (D302R +R306E)	5′ GTTAGAATGGTGGCCGAACCGTCCATGGGTCTT 3′	5′ AAGACCCATGGACGGTTCGGCCACCATTCTAAC 3′
MCT1 R404E	5′ CCACTTTTAGGCGAGCTCAATGACATGTATGG 3′	5′ CCATACATGTCATTGAGCTCGCCTAAAAGTGG3′
Basigin E218R	5′ CTGGGCATTGTGGCCAGGGTCCTGGTGTTGGTC 3′	5′ GACCCGTAACACCGGTCCCAGGACCACAACCAG 3′
Basigin E218Q	5′ GGGCATTGTGGCCCAGGTCCTGGTGTTG 3′	5′ CAACACCAGGACCTGGGCCACAATGCCC 3′

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 µg 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.

Table I.  Summary of the expression and transport activity of wild-type and mutant MCT1 expressed in Xenopus laevis oocytes in the presence and absence of WT- and mutant basigin. Where given, V_max and K_m values (±SE) were determined from the initial rates of transport of L-lactate determined using BCECF fluorescence as described under Methods. Four oocytes were used in each case and L-lactate was added at 2.5, 5, 10, 20 and 40 mM. Data analysis was performed as described previously (Manning Fox et al. [2000]). Where no transport could be detected using BCECF confirmation was made by determining the uptake of 0.5 mM L-[¹⁴C]-lactate at 10 min as described under Methods. Transport was indicated when uptake was significantly greater than in water injected oocytes, but in no case in which no BCECF response was detected was this observed. nd: not determined.

	Transport measured using BCECF			Plasma membrane expression determined using:
Oocyte injection	Detectable	Km±SE (mM)	Vmax±SE (▵F.min^−1)	Western blot	Sections
WT-MCT1	Yes	3.52±0.23	0.42±0.007	Yes	Yes
R306E-MCT1	No	–	–	No	No
D302R/R306E-MCT1	No	–	–	Yes	Yes
R306K-MCT1	No	–	–	Yes	Yes
WT-MCT1 + WT-basigin	Yes	2.94±0.16	0.36±0.005	Yes	Yes
WT-MCT1 + E218Q-basigin	Yes	2.73±0.21	0.48±0.009	Yes	Yes
R306E-MCT1 + E218R-basigin	No	–	–	No	No
D302R/R306E-MCT1 + E218R-basigin	No	–	–	Yes	Yes
R306K-MCT1 + E218R-basigin	No	–	–	Yes	Yes
R86Q-MCT1	Yes	8.79±1.5	0.42±0.034	Yes	nd
R86E-MCT1	Yes	6.48±1.5	0.67±0.050	Yes	nd

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 co-transfected 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.

Supplementary Table II. The effects of mutating charged residues in potential TM helices of MCT1 on its plasma membrane expression and activity. Data are summarized from previous work of others (Rahman et al. 1999, Galic et al. 2003) and the present studies.

Mutation in rat MCT1	Position in Human MCT1	TM or loop	Plasma membrane expression	Activity	Laboratory
D15N	D15	1	Yes	Yes	Galic et al. 2003
K38Q	K38	1	Yes	Yes	Present paper
K45Q	K45	1	Yes	Yes	Present paper
R86Q	R86	3	Yes	Yes	Present paper
R86E	R86	3	Yes	Yes	Present paper
K137Q	K137	4–5	Yes	Yes	Galic et al. 2003
K141Q	K141	4–5	Yes	Yes	Galic et al. 2003
K142Q	K142	4–5	Yes	Yes	Galic et al. 2003
R143Q	R143	5	Yes	No	Galic et al. 2003
R143H	R143	5	Yes	Yes	Galic et al. 2003
R143K	R143	5	Yes	No	Galic et al. 2003
R143E	R143	5	Yes	No	Galic et al. 2003
R143E/E369R	R143	5/10	Yes	No	Galic et al. 2003
R175Q	R175	5–6	Yes	Yes	Present paper
R196E	R196	6	Yes	Yes	Present paper
R196Q	R196	6	Yes	Yes	Present paper
K204E	K204	6, 6–7	Yes	Yes	Present paper
K282Q	K289	7–8	Yes	Yes	Present paper
K282R	K289	7–8	Yes	Yes	Present paper
K290Q	K297	7–8	Yes	Yes	Present paper
K290R	K297	7–8	Yes	Yes	Present paper
D302E	D309	8	Yes	No	Rahman et al. 1999
D302N	D309	8	No	No	Rahman et al. 1999
R306T	R313	8	Low	?	Rahman et al. 1999
R306E	R313	8	No	No	Present paper
D302R/R306E	D309R/R313E	8	Yes	No	Present paper
H337Q	H344	9	Yes	Yes	Rahman et al. 1999
E369R	E376	10	Yes	No	Rahman et al. 1999
E391Q	E398	11	Yes	Yes	Rahman et al. 1999
R404E	R410	11	Yes	Yes	Present paper
K413R	K420	11–12	Yes	Yes	Present paper
K413Q	K420	11–12	Yes	Yes	Present paper

Manning Fox JE, Meredith D, Halestrap AP. Characterisation of human monocarboxylate transporter 4 substantiates its role in lactic acid efflux from skeletal muscle. J Physiol 2000; 529: 285–293

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