4F2hc/LAT1 heterodimer: a promising candidate for solving the first atomic structure of a heteromeric amino acid transporter = Heterodímero 4F2hc/LAT1: un candidato prometedor para resolver la primera estructura atómica de un transportador heteromérico de aminoácidos

Abstract

Heteromeric amino acid transporters (HATs) are the unique solute transporters known to be composed of two subunits linked by a disulphide bridge. The heavy subunit (4F2hc or rBAT), a type II membrane N-glycoprotein that traffics HATs to the plasma membrane, and a light subunit, a polytopic membrane protein of the L-Amino acid transporters family (LATs), that is the catalytic subunit. HATs are involved in several physiological processes to sustain life, by carrying amino acids to the places in which they are required for cellular growing, metabolism and signaling pathways. For this reason, overexpression of some of these HATs or their dysfunction is related to different diseases, like inherited aminoacidurias, tumor growth, viral infection and cocaine relapse, among other. Thus, structure determination of these HATs would be very valuable to understand the molecular mechanism and function underlying HATs amino acid transport and their related diseases, which could be useful to develop therapeutic drugs. Beside this relevance, there is no a single structure solved of a LAT, and the structural information concerning interactions between heavy and light subunits is very scarce, due to the challenging of expressing at enough yield and stability eukaryotic membrane proteins for structural studies.

In the present work, the stability and dispersity in solution of three different vertebrate HATs, whose LATs have been previously found to be the most stable among 24 metazoan LATs, were investigated to find a good candidate for structural studies. We found that vertebrate 4F2hc/LAT1 was the most stable among the three HATs, and that the expression of such heterodimer in Pichia pastoris increased the yield of protein obtained in comparison with insect cells. This heterodimer showed significantly increased stability respect to human 4F2hc/LAT2, whose structural model at low resolution (21 Å) was determined by 3D reconstruction from negatively stained complexes, in our lab. Thus, evidence corroborating that the stability of HATs are directly involved with the stability of the light subunit, and with the stabilizing effect of 4F2hc heavy chain on those LATs, is provided here.

4F2hc/LAT1, and mainly its light subunit (LAT1), showed binding of substrate and of human LAT1 inhibitor, KYT-0353, after being solubilized and purified in DDM detergent plus a derivative of cholesterol (CHS). This suggests that these proteins purified in DDM/CHS from Pichia membranes are active and stable, and that vertebrate 4F2hc/LAT1 would offer a very approximate structural model to human HATs. On the other hand, 4F2hc/LAT1 could be obtained relatively stable and at enough yield when solubilized in an amphipatic polymer, widely used for structural studies of eukaryotic membrane proteins by cryo-EM, amphipol A8-35. Finally, the 3D reconstruction at low resolution of 4F2hc/LAT1 solubilized in A8-35, reported here, indicates that amphipol solubilization increases the structural definition of the 4F2hc/LAT1 respect to the observed in the previous models of human 4F2hc/LAT2 solubilized in detergent and CHS, probably because the elimination of the DDM bound to the transmembrane segments, and also because of the use of a more stable heterodimer. Taken together, these results indicate that 4F2hc/LAT1 is a good candidate for structural studies by cryo-EM, also due to the recent advances for imaging molecules of close molecular weight to 4F2hc/LAT1.

Los transportadores heteroméricos de aminoácidos (HATs) están constituidos por una subunidad pesada (4F2hc o rBAT), y una subunidad ligera, unidas por un puente disulfuro. La subunidad pesada es una N-glicoproteína de tipo II que localiza los HATs en la membrana plasmática, mientras que la subunidades ligeras son proteínas politópicas de la familia de los LATs, siendo las subunidades catalíticas. Debido a la relevancia y variedad de funciones en las cuales los aminoácidos son requeridos, estos HATs están involucrados en gran cantidad de procesos fisiológicos, al igual que en enfermedades, como consecuencia de su sobre expresión, o de la disfunción de algunos de ellos. Por esto, es importante obtener su estructura para entender su mecanismo molecular y función, así como su relación con las enfermedades asociadas, como son las aminoacidurias hereditarias y el cáncer, entre otras. Sin embargo, debido a la baja estabilidad y producción de este tipo de proteínas, no se ha resuelto la estructura de ningún LAT, por lo cual también falta información acerca de la interacción entre la subunidad pesada y la ligera y su implicación en el transporte. En el presente trabajo, se encontró que 4F2hc/LAT1 de vertebrado es un buen candidato para realizar estudios estructurales usando cryo-EM. Este heterodímero fue obtenido en suficiente cantidad en Pichia pastoris, y mostró buena estabilidad cuando fue solubilizado y purificado en detergente DDM, más el derivado del colesterol, CHS, y en amphipol A8-35, un polímero anfipático ampliamente usado en estudios estructurales de proteínas de membrana por cryo-EM. 4F2hc/LAT1, pero más claramente su subunidad ligera (LAT1), se mostraron activos después de ser purificados en DDM/CHS, mostrando unión a sustrato y al inhibidor de LAT1 humano KYT-0353. Finalmente, un modelo a baja resolución de 4F2hc/LAT1 solubilizado en A8-35, mostró que la solubilización en amphipol, así como la utilización de un heterodímero más estable, mejoró la definición estructural del mapa 3D, en comparación con modelos anteriores a baja resolución de 4F2hc/LAT2 humano solubilizado en detergente y CHS.