title: State-dependent conformations of the translocation pathway in the tyrosine transporter Tyt1, a novel neurotransmitter:sodium symporter from Fusobacterium nucleatum.
abstract: The gene of a novel prokaryotic member (Tyt1) of the neurotransmitter:sodium symporter (NSS) family has been cloned from Fusobacterium nucleatum. In contrast to eukaryotic and some prokaryotic NSSs, which contain 12 transmembrane domains (TMs), Tyt1 contains only 11 TMs, a characteristic shared by approximately 70% of prokaryotic NSS homologues. Nonetheless upon heterologous expression in an engineered Escherichia coli host, Tyt1 catalyzes robust Na+-dependent, highly selective l-tyrosine transport. Genetic engineering of Tyt1 variants devoid of cysteines or with individually retained endogenous cysteines at positions 18 or 238, at the cytoplasmic ends of TM1 and TM6, respectively, preserved normal transport activity. Whereas cysteine-less Tyt1 was resistant to the inhibitory effect of sulfhydryl-alkylating reagents, N-ethylmaleimide inhibited transport by Tyt1 variants containing either one or both of the endogenous cysteines, and this inhibition was altered by the substrates sodium and tyrosine, consistent with substrate-induced dynamics in the transport pathway. Our findings support a binding model of Tyt1 function in which an ordered sequence of substrate-induced structural changes reflects distinct conformational states of the transporter. This work identifies Tyt1 as the first functional bacterial NSS member putatively consisting of only 11 TMs and shows that Tyt1 is a suitable model for the study of NSS dynamics with relevance to structure/function relationships of human NSSs, including the dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid transporters.
authors.affiliation: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yano
authors.forename: Naomi R
authors.initials: NR
authors.lastname: Goldberg
authors.forename: Lihua
authors.initials: L
authors.lastname: Duan
authors.forename: Thijs
authors.initials: T
authors.lastname: Beuming
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D016967:Fusobacterium nucleatum / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018377:Neurotransmitter Agents / Q000378:metabolism; D010802:Phylogeny; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism*; D027981:Symporters / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018377:Neurotransmitter Agents; D050480:Plasma Membrane Neurotransmitter Transport Proteins; D027981:Symporters; D014443:Tyrosine; D012964:Sodium; D003545:Cysteine