Fusion of human monocarboxylate transporter 1 with basigin and expression in S. cerevisiae : Is basigin more than a chaperone?
Lactate is a key metabolite in human cells. A regulated transport across membranes is vital for cellular function while deregulated transport is a hallmark of cancer. In tumor cells, glycolysis as the main route for ATP synthesis even in the presence of oxygen (Warburg effect) demands rapid lactate clearance to avoid acidification. Oxygenic cancer cells, in turn, rely on an efficient retrieval of extracellular lactate to fuel the citric acid cycle (reverse Warburg effect). To maintain an active interchange between cells, four monocarboxylate transporters (MCT1−4) manage bi-directional, proton-coupled transport across plasma membranes. For its translocation to the plasma membrane, MCT1 demands chaperoning by a member of the immunoglobulin superfamily, namely basigin. Both proteins remain complexed at the plasma membrane. Although frequently suggested, a direct effect of the chaperone on MCT1-mediated transport is not resolvable in commonly used expression systems. In this study, MCT1 expression in S. cerevisiae Δjen1 Δady2 profited from a basigin-independent translocation in a system with zero background from endogenous monocarboxylate transporters or basigin homologs. The molecular fusion with truncated basigin constructs revealed an effect on transmembrane L-lactate distribution at the domain level. In zero-trans influx experiments using 14C-labeled substrate, the presence of basigin’s extracellular Ig-I domain permitted a 4.5-fold intracellular L-lactate accumulation in the transport equilibrium. At near-neutral pH, cytosolic L-lactate concentrations greatly exceeded those provided with the buffer. The absence of the basigin Ig-I domain due to truncation or misfolding reversed this effect. The identification of patches of positive and negative surface potentials and evidence from charge-resolving point mutations indicated an electrostatic attraction of L-lactate anions and protons. This thesis deduces a substrate harvesting function of basigin that creates a “microenvironment” of locally increased concentrations and drives L-lactate influx according to Le Chatelier’s principle. This influx was physiologically relevant and promoted cell growth on L-lactate medium. According to classical and reverse Warburg effects, highly adapted tumor cells require a fine-tuned transmembrane L-lactate distribution and basigin might be an important determinant. Hereof, MCTs are promising targets in the anti-tumor therapy. The basigin-MCT1 fusion set-up from this study further revealed two known MCT1 inhibitors, AZD3965 and p-chloromercuribenzene sulfonate (pCMBS), as direct and basigin-independent modifiers. Cys159 in the transporter cavity was revealed as selectively targetable by pCMBS leading to a complete transport inhibition. Smaller cysteine-modifiers had a less prominent effect and lacked site-specificity. Cys159 is proposed to constitute a hinge region of the alternating access transporter and a wedge-like modification locks MCT1 in the outward open conformation. This reveals a target region for inhibitor design and in the future, Cys159 might serve as a natural anchor to introduce distinct labels and report on physicochemical modalities in a most critical part of the transporter.
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