title: Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol.
abstract: LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmaxbut not the Kmof the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold.
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK.
authors.forename: David
authors.initials: D
authors.lastname: Dickens
authors.affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, UK.
authors.forename: George N
authors.initials: GN
authors.lastname: Chiduza
authors.affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, UK.
authors.forename: Gareth S A
authors.initials: GS
authors.lastname: Wright
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK.
authors.forename: Munir
authors.initials: M
authors.lastname: Pirmohamed
authors.affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, UK.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Antonyuk
authors.affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, UK.
authors.forename: S Samar
authors.initials: SS
authors.lastname: Hasnain
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D027261:Fusion Regulatory Protein-1 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D027283:Large Neutral Amino Acid-Transporter 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007980:Levodopa / Q000378:metabolism / Q000494:pharmacology; D050356:Lipid Metabolism / Q000187:drug effects; D008563:Membrane Lipids / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs
chemical_list: D000596:Amino Acids; D027261:Fusion Regulatory Protein-1; D027283:Large Neutral Amino Acid-Transporter 1; D008563:Membrane Lipids; D007980:Levodopa; D002784:Cholesterol