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J. Biol. Chem., Vol. 281, Issue 36, 26444-26454, September 8, 2006

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State-dependent Conformations of the Translocation Pathway in the Tyrosine Transporter Tyt1, a Novel Neurotransmitter:Sodium Symporter from Fusobacterium nucleatum^*

Matthias Quick, Hideaki Yano, Naomi R. Goldberg, Lihua Duan, Thijs Beuming, Lei Shi, Harel Weinstein^¶, and Jonathan A. Javitch^||1

From the Center for Molecular Recognition and ^||Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032 and Department of Physiology and Biophysics and ^¶His Royal Highness Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York 10021

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 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 -aminobutyric acid transporters.

Received for publication, March 15, 2006 , and in revised form, May 18, 2006.

^* This work was supported by National Institutes of Health Grants MH57324 and DA17293 (to J. A. J.) and DA12408 (to H. W. and J. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, P&S 11-401, Box 7, New York, NY 10032. Tel.: 212-305-7308; Fax: 212-305-5594; E-mail: jaj2{at}columbia.edu.

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