Abstract by Maria Rafiq

Cellular transport which can be mediated by different transport mechanisms, is vital for homeostasis. The major facilitator superfamily (MFS) of secondary active transporters plays a significant role in the translocation of compounds across the impermeable cell membrane. In this thesis, I focus on two MFS families, the proton-coupled oligopeptide transporters (POTs) and monocarboxylate transporters (MCTs), more specifically, hPEPT1 and MCT1.

POTs are integral membrane proteins that facilitate the uptake of peptides and peptidomimetics into the cell, and they also have an essential role in drug transport as well. In the first study, we developed an optimized protocol for the expression and purification of full-length human proton-coupled oligopeptide transporter 1 (hPEPT1). The transport function of the overexpressed protein was established using both intact cells and membrane preparations. Detergent-solubilized hPEPT1 was found to be stable at 71°C and pH-optimum being 6.0. The purified protein was able to bind peptides under these conditions. Size-exclusion studies showed both monomer and dimer as well as possibly bigger species. Negative stain electron microscopy revealed a mixture of species both in lauryl maltose neopentyl glycol and n-dodecyl-β-D-maltoside, although monomers were dominant. Monomeric hPEPT1 2D classes showed the ectodomain and the transmembrane region embedded in the micelle.

MCTs facilitate the transport of monocarboxylate across cell membranes. Human MCTs perform several physiological functions and are widely expressed in the body. BSG-2 (BSG2), a chaperone protein, stabilizes MCT1 at the membrane by direct interaction. In this study, we established the production of the recombinant human MCT1 and BSG2 complex as well as full-length BSG2 alone and showed that in both preparations BSG2 can interact with the malarial protein Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5). Interestingly, we saw that the MCT1-BSG2 complex can cross-link with glutaraldehyde in the absence of RH5, but not in the presence of RH5. This implies that interaction between RH5 and BSG2 results in conformational changes in the MCT1-BSG2 complex leading to the instability of the ternary complex.