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J. Biol. Chem., Vol. 280, Issue 34, 30638-30647, August 26, 2005

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Identification of Conserved Prolyl Residue Important for Transport Activity and the Substrate Specificity Range of Yeast Plasma Membrane Na⁺/H⁺ Antiporters^*

Olga Kinclova-Zimmermannova, Martin Zavrel, and Hana Sychrova

From the Department of Membrane Transport, Institute of Physiology AS CR, Prague 4, 142 20, Czech Republic

Yeast plasma membrane Na⁺/H⁺ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K⁺), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K⁺) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Site-directed mutagenesis of Pro¹⁴⁵ proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K⁺. Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na⁺ and Li⁺. Replacement of Pro¹⁴⁵ with glycine resulted in a ZrSod2-22p with extremely low activity only for Na⁺, and the exchange of a charged residue (Asp, Lys) for Pro¹⁴⁵ completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro¹⁴⁶) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.

Received for publication, June 10, 2005

^* This work was supported by Czech Grant Agencies (GA CR 204/02/D092, AV0Z 5011922). 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.

To whom correspondence should be addressed: Dept. of Membrane Transport, Institute of Physiology AS CR, Videnska 1083, Prague 4, 142 20, Czech Republic. Tel.: 420-241-06-26-73; Fax: 420-241-06-24-88; E-mail: kinclova{at}biomed.cas.cz.

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This article has been cited by other articles:

				O. Kinclova-Zimmermannova and H. Sychrova Plasma-membrane Cnh1 Na+/H+ antiporter regulates potassium homeostasis in Candida albicans Microbiology, August 1, 2007; 153(8): 2603 - 2612. [Abstract] [Full Text] [PDF]