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 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 basigin as well as the cRNA for WT-, E218Q- or E218R-basigin was co-injected with the MCT1 cRNA.<p><b>Copyright information:</b></p><p>Taken from "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"</p><p></p><p>Molecular Membrane Biology 2006;23(6):486-498.</p><p>Published online 13 Dec 2006</p><p>PMCID:PMC2409183.</p><p></p

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 online.<p><b>Copyright information:</b></p><p>Taken from "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"</p><p></p><p>Molecular Membrane Biology 2006;23(6):486-498.</p><p>Published online 13 Dec 2006</p><p>PMCID:PMC2409183.</p><p></p

FRET measurements suggest that mutation of Arg 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.<p><b>Copyright information:</b></p><p>Taken from "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"</p><p></p><p>Molecular Membrane Biology 2006;23(6):486-498.</p><p>Published online 13 Dec 2006</p><p>PMCID:PMC2409183.</p><p></p

Mutation of Arg or Arg 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 online.<p><b>Copyright information:</b></p><p>Taken from "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"</p><p></p><p>Molecular Membrane Biology 2006;23(6):486-498.</p><p>Published online 13 Dec 2006</p><p>PMCID:PMC2409183.</p><p></p

R306K-MCT1 is expressed at the plasma membrane of Xenopus oocytes but is inactive

Details are as given for . 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 online.<p><b>Copyright information:</b></p><p>Taken from "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"</p><p></p><p>Molecular Membrane Biology 2006;23(6):486-498.</p><p>Published online 13 Dec 2006</p><p>PMCID:PMC2409183.</p><p></p

D302R/R306E-MCT1 is expressed at the plasma membrane of Xenopus oocytes but is inactive

Details are as given for . 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 online.<p><b>Copyright information:</b></p><p>Taken from "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"</p><p></p><p>Molecular Membrane Biology 2006;23(6):486-498.</p><p>Published online 13 Dec 2006</p><p>PMCID:PMC2409183.</p><p></p

MCT2 immunoreactivity in 40 µm sagittal sections through the adult rat cerebellar cortex reveal Purkinje cell immunoreactivity.

<p><b>A:</b> MCT2 immunoreactivity visualised by peroxidase reaction product is deposited in the Purkinje cell dendrites in the molecular layer (ml), Purkinje cell somata in the Purkinje cell layer (pcl), and weakly in the granular layer (gl). <b>B:</b> Anti-MCT2 immunofluorescence staining is prominent in the granular layer (gl). <b>C:</b> Anti-MCT2 immunofluorescence staining outlines Purkinje cell somata in the Purkinje cell layer (pcl) and their proximal dendrites in the molecular layer (ml): the distal dendritic arbour is either weakly stained or unreactive. Scale bar = 50 µm.</p

Co-transfection with np55 or np65 enables expression of MCT2 at the plasma membrane.

<p>Cos-7 cells were transiently transfected with MCT2 tagged at its N-terminus with CFP and/or np55 or np65 C-terminally labelled with YFP. The expression of np55 or np65 and MCT2 was detected by confocal microscopy. <b>A:</b> In single transfected cells MCT2 remains in the perinuclear region while neuroplastin is located at the plasma membrane and in the soma. <b>B and C:</b> In contrast, when MCT2 is transfected together with np55 or np65 both proteins are expressed at the cell surface. Cells were imaged using the Leica confocal imaging spectrophotometer system (TCS-SP2) attached to a Leica DMIRBE inverted epifluorescence microscope.</p

MCT2 is selectively expressed by Purkinje cell subsets.

<p><b>A:</b> Levels of MCT2 immunoreactivity vary between Purkinje cell somata of the Purkinje cell layer (pcl) from strong (white arrowhead), to weak (black arrow), to none (black arrowhead). No boundaries are seen in the granular layer (gl). <b>B:</b> Clear boundaries are seen in the molecular layer (ml) between regions that express high levels of MCT2 immunoreactivity and others that are either weakly reactive or unreactive (arrows). <b>C:</b> A transverse section through lobule VIa of the adult rat cerebellar cortex double immunofluorescence labelled for zebrin II/aldolase C (green) and MCT2 (red), showing an array of alternating Purkinje cell stripes. The stripes of MCT2 immunoreactivity alternate with those of zebrin II/aldolase C. Scale bars: A and C = 50 µm, B = 100 µm.</p

<i>Xenopus</i> oocytes express neuroplastin that facilitates MCT2 expression at the plasma membrane.

<p><b>A:</b> shows the results of RT-PCR performed on mRNA extracted from <i>Xenopus laevis</i> oocytes, liver and thymus as described in the Experimental section. Primers for basigin (Bas), embigin (Emb) and neuroplastin (Np) were used as indicated. <b>B:</b> shows the expression of MCT2 in oocytes with or without antisense mRNA for basigin or neuroplastin detected by immunofluorescence microscopy. Antisense treatment reduces expression at the plasma membrane (PM) but increases intracellular expression (IC). The scale bar is 50 µm. <b>C:</b> shows parallel data for MCT2 expression in a crude membrane fraction derived from the oocyte and detected by SDS-PAGE and Western blotting. Here, scrambled (Scram) antisense RNA is used as an additional control. <b>D:</b> presents parallel data for the transport of [¹⁴C]-L-lactate into oocytes determined over 5 min. Error bars represent the S.E.M. of the number of oocytes indicated.</p