Determination of external loop topology in the serotonin transporter by site-directed chemical labeling.
The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1, 3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine. These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEA-biotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and gamma-aminobutyric acid transporters.
Chen
J G
JG
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510-8066, USA.
Liu-Chen
S
S
Rudnick
G
G
D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D050486:Serotonin Plasma Membrane Transport Proteins; D013388:Succinimides / Q000737:chemistry
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D013388:Succinimides; C094773:sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; D001710:Biotin; D008239:Lysine; D003545:Cysteine