AbstractsBiology & Animal Science

Heteromeric amino acid transporters (HATs) mediate the transport of amino acids through the plasma membrane. They are composed of two subunits (a heavy and a light one) linked by a conserved disulfide bridge. Genetic defects in the genes coding these HATs may affect its functionality or expression, leading to inherited aminoacidurias. Thus, solving the structure of the Eukaryotic HATs has become of great importance. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, human 4F2hc/L-type amino acid transporter 2 (LAT2) first low resolution 3D model obtained by single particle negative-staining transmission electron microscopy (TEM) was validated. In order to assess the interaction between both subunits of the heterodimer, crosslinking experiments between cysteine residues in both moieties was tried. Namely, two chemical spacers of different length (10.5 and 14.3 Å) were tested and crosslinking was observed for those mutants with pairing positions between 8 and 17.5 Å. Indeed, specific residues that crosslinked 4F2hc and LAT2 nearly completely (>80%). As a result of the positive results (as compared to the appropriate controls) the idea that 4F2hc-ED almost completely covers the extracellular surface of the transporter subunit LAT2 is reasonable. Moreover, further varied evidences (TEM, SPA and docking experiments) were in line with the obtained results, revealing that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs. In addition, it has been demonstrated that the ectodomain of 4F2hc suffices the stabilization of the light subunit. The second goal of the thesis was to find a suitable HAT candidate to perform crystallization trials and posterior structure elucidation, since human 4F2h/LAT2 was not stable enough for this aim. Until now, only the human 4F2hc ectodomain atomic structure has been solved (Fort et al., 2007), and some low sequence amino acid identity prokaryotic homologues of LATs. In order to identify putative good eukaryotic light subunits for 3D crystallization the adopted GFP‐based Saccharomyces cerevisiae protocol for our transporters resulted successful since it allowed to find three putative good candidate eukaryotic light subunits for 3D crystallization studies. GFP technology allowed quick expression screening, membrane protein-detergent solubilization screening and finally another screening step including assessment of the stability by ultracentrifugation dispersity sedimentation. Once the candidates selected in the best conditions, further purification was required (size exclusion chromatography) before attempting crystallization. In this sense, further efforts were delivered in order to try to enhance the stability (and minimize aggregation). Thus, addition of lipids in the solubilization…