Key resources table.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Aquifex aeolicus)	LeuT gene	https://doi.org/10.1038/ncomms12755
Strain, strain background (Escherichia coli)	BL21(DE3)	Sigma-Aldrich	CMC0016	Electrocompetent cells
Strain, strain protein expression (Escherichia coli)	C41(DE3)	Sigma-Aldrich	CMC0017	Cells for protein expression
Recombinant DNA reagent	LeuT; LeuT WT (plasmid)	https://doi.org/10.1038/ncomms12755		pET16b backbone
Recombinant DNA reagent	LeuTtmFRET; LeuT A313H-A317H-K398C (plasmid)	https://doi.org/10.1038/ncomms12755		pET16b backbone
Recombinant DNA reagent	LeuTK398C; LeuT K398C (plasmid)	https://doi.org/10.1038/ncomms12755		pET16b backbone
Sequence-based reagent	Primers for generation of LeuT mutants	This paper		See list of primers in Appendix. Primers were synthesized by Eurofins Genomics
Chemical compound, drug	fluorescein-5-maleimide; F5M	Thermo Fischer Scientific	F150
Chemical compound, drug	[3H]leucine	PerkinElmer	NET1166001
Chemical compound, drug	E. coli polar lipid extract	Avanti Polar Lipids	100600 C
Chemical compound, drug	[3H]alanine	Moravek Biochemicals		Made on request
Chemical compound, drug	Gel stain InstantBlue	abcam	Ab119211
Software, algorithm	GraphPad Prism 9.0	GraphPad Software, Boston, Massachusetts USA
Other	Yttrium Silicate Copper (YSi-Cu) His-tag SPA beads; YSi-Cu His-tag SPA beads	PerkinElmer	RPNQ0096	SPA beads used for scintillation proximity assays - See: Pharmacological characterization of LeuT mutants. In Materials & Methods Section
Other	thrombin	Cytiva	27084601	Protease used to cleave the His-tag from LeuT
Other	Filtermat B – GF/B	PerkinElmer	1450–521	Filters used to trap proteoliposomes for scintillation counting of [3 H]ligand
Other	MeltiLex B/HS	PerkinElmer	1450–442	Scintillation plates to add on Filtermats for [3 H] counting
Other	Nuclepore Track-Etch Membrane polycarbonate filter of pore size 400 nm	Sigma-Aldrich	WHA10417104	For extrusion of PLs.
Other	SM-2 Bio-Beads	Bio-Rad Laboratories	1523920	For detergent extraction from the specimen to promote reconstitution of LeuT into PLs