Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin.
Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.
Université Paris Descartes, Sorbonne Paris Cité, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8192, Centre Universitaire des Saints-Pères, 45 rue des Saints-Pères, F-75006 Paris, France.
Raquel
R
Ruivo
Gian Carlo
GC
Bellenchi
Xiong
X
Chen
Giovanni
G
Zifarelli
Corinne
C
Sagné
Cécile
C
Debacker
Michael
M
Pusch
Stéphane
S
Supplisson
Bruno
B
Gasnier
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D026921:Amino Acid Transport Systems, Neutral; C111445:CTNS protein, human; D011522:Protons