The genomic structure of the human lactate transporters MCT1 and MCT2 and evidence for alternative promoters

Abstract

Tethered bilayers are formed on solid supports by the interaction of lipid vesicles with mixed self-assembled monolayers (SAMs). The mixed monolayer assembles on a gold support in a solution of hydrophilic and hydrophobic thiols producing an amphipathic surface of the two components. The hydrophobic molecules are composed of an ethoxythiol tether and a cholesterol head group. Lipid vesicles spontaneously unroll on the mixed surface forming a tethered bilayer ; the hydrophobic head group anchoring it to the support and the hydrophilic spacer molecule maintaining a water layer underneath. In this poster, monolayers of different molecular compositions are characterised in terms of the physical, kinetic and electrical properties of the bilayer. SAMs with different cholesterol content, ethoxythiol tether length and phospholipid head groups are shown. The tethered bilayer system is used to study rhodopsin photoactivation. Activation of rhodopsin causes the protein, transducin, to leave the membrane. Rhodopsin-containing membranes are isolated from retinal tissue, and used to form a tethered bilayer in the dark. A flash of light activates the protein, causing the exit of transducin from the membrane, which can be observed by surface plasmon resonance, demonstrating the extent to which a tethered bilayer resembles a biological membrane. 52 Confirmation of the interaction between the monocarboxylate transporter MCTl and its ancillary protein CD147 using fluorescence resonance energy transferWe have shown previously that the monocarboxylate transporter (MCT1) is tightly associated with the monotopic membrane glycoprotein CD147. This protein is required for the proper expression of both MCTl and MCT4 at the plasma membrane.Here we describe the expression in COS cells of constructs of MCTl and CD147 in which cyan-and yellow-fluorescent protein (CFP and YFP respectively) have been attached to either the Cor N-terminal ends of both proteins. These constructs, like the native proteins, were properly targeted to the plasma membrane only when co-expressed and then enhanced lactic acid transport into the cells 3-4 fold. Fluorescence energy transfer (FRET) between the two proteins was strong only when both fluorophores were present on the cytosolic face of the proteins. A range of controls including photobleaching experiments were used to confirm that the FRET signal was not due to either excitation of the YFP by the CFP excitation light or by overcrowding. 53The genomic structure of the human lactate transporters