ATP modulates SLC7A5 (LAT1) synergistically with cholesterol

Jessica Cosco,Michele Galluccio,Cesare Indiveri,Filomena Rovella,Tiziano Mazza,Gerhard F. Ecker,Mariafrancesca Scalise,Claire Colas,Riccardo Martini

doi:10.1038/s41598-020-73757-y

Abstract

The plasma membrane transporter hLAT1 is responsible for providing cells with essential amino acids. hLAT1 is over-expressed in virtually all human cancers making the protein a hot-spot in the fields of cancer and pharmacology research. However, regulatory aspects of hLAT1 biology are still poorly understood. A remarkable stimulation of transport activity was observed in the presence of physiological levels of cholesterol together with a selective increase of the affinity for the substrate on the internal site, suggesting a stabilization of the inward open conformation of hLAT1. A synergistic effect by ATP was also observed only in the presence of cholesterol. The same phenomenon was detected with the native protein. Altogether, the biochemical assays suggested that cholesterol and ATP binding sites are close to each other. The computational analysis identified two neighboring regions, one hydrophobic and one hydrophilic, to which cholesterol and ATP were docked, respectively. The computational data predicted interaction of the ϒ-phosphate of ATP with Lys 204, which was confirmed by site-directed mutagenesis. The hLAT1-K204Q mutant showed an impaired function and response to ATP. Interestingly, this residue is conserved in several members of the SLC7 family.

Highlights

The human LAT1 transporter (SLC7A5) is one of the seven members of the SLC7 family characterized by the association with ancillary glycoproteins belonging to the SLC3 family, namely SLC3A1 and SLC3A21,2
In good agreement with this postulate, it has been recently demonstrated, by experiments conducted on mice and in vitro models, that two natural point mutations of LAT1 are responsible for the appearance of a familiar form of autism spectrum disorders (ASD) characterized by a lower supply of essential amino acids in brain[5]
The role of cholesterol in modulating the transport activity of hLAT1 is described in this work

Summary

Introduction

The human LAT1 transporter (SLC7A5) is one of the seven members of the SLC7 family characterized by the association with ancillary glycoproteins belonging to the SLC3 family, namely SLC3A1 and SLC3A21,2 The formation of these heterodimers represents a peculiar feature for mammalian transporters in terms of both structural and functional properties. LAT1 has a quite narrow tissue distribution: it is mainly expressed in placenta and the blood–brain barrier In these districts, the supply of essential amino acids is fundamental for normal cell growth and d evelopment[2]. In the present work, we aimed to investigate the modulation of LAT1 transport activity by physiological effectors, employing the experimental model of proteoliposomes This tool gives the possibility of modifying the lipid composition of the membrane as well as precisely controlling the composition of the external and internal aqueous compartments of the protein-harboring v esicles[19]. The molecular basis of the pH sensitivity was described, moving a step forward in completing the knowledge on human LAT1

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