Abstract
Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme's intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.
Highlights
Many tumor cells, especially those that reside in a hypoxic environment, rely on glycolysis to meet their increased demand for energy and biosynthetic precursors (Hanahan and Weinberg, 2011; Schulze and Harris, 2012; Parks et al, 2013)
We have recently shown that extracellular CAIX facilitates lactate transport in hypoxic cancer cells by acting as a proton antenna for MCT1/4 at the extracellular face of the plasma membrane (Jamali et al, 2015)
As proton-driven lactate transport would benefit from a proton antenna at the cytosolic face of the cell membrane, this work investigated whether proton–lactate co-transport in cancer cells is facilitated by intracellular CAII
Summary
Especially those that reside in a hypoxic environment, rely on glycolysis to meet their increased demand for energy and biosynthetic precursors (Hanahan and Weinberg, 2011; Schulze and Harris, 2012; Parks et al, 2013). The first evidence that this non-catalytic interaction between MCT1/4 and CAII requires direct binding between the two proteins was provided by injection of CAII that was bound to an antibody prior to the injection In this experiment, CAII was not able to enhance the transport activity of MCT1 in Xenopus oocytes, suggesting a sterical suppression of the interaction by the antibody (Becker and Deitmer, 2008). By introduction of single site mutations in MCT1 and MCT4, the glutamic acidic clusters E489EE and E431EE within the C-terminal tail of MCT1 and MCT4, respectively, could be identified as binding sites for CAII (Stridh et al, 2012; Noor et al, 2015) In both studies, binding was confirmed both on the functional level by measuring MCT transport activity in Xenopus oocytes and by pull-down assays using GST-fusion proteins. Our results showed that CAII features a moiety (Glu69 + Asp72) that exclusively mediates proton exchange with the transporter, while the central residue of its intramolecular proton shuttle (His64) mediates the binding of CAII to MCT1 and MCT4
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