ABSTRACT

Substrate binding sites in Kdp, a P-type ATPase of Escherichia coli, were identified by the isolation and characterization of mutants with reduced affinity for K, its cation substrate. Most of the mutants have an altered KdpA subunit, a hydrophobic subunit not found in other P-type ATPases. Topological analysis of KdpA and the locations of the residues changed in the mutants suggest that KdpA has 10 membrane-spanning segments and forms two separate and distinct sites where K is bound. One site is formed by three periplasmic loops of the protein and is inferred to be the site of initial binding. The other site is cytoplasmic. We believe K moves from the periplasmic site through the membrane to the cytoplasmic site where it becomes ``occluded,'' i.e. inexchangeable with K outside the membrane. Membrane-spanning parts of KdpA probably form the path for transmembrane movement of K. The kinetics of cation transport in the mutants indicate that each of the two binding sites contributes to the observed K for cations as well as to the marked discrimination between K and Rb characteristic of wild-type Kdp. Energy coupling in Kdp, mediated by the KdpB subunit, is performed by a different subunit from the one that mediates transport.

FOOTNOTES

*

This work was supported by Grant GM22323 from NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Molecular and Cell Biology, University of Aberdeen, AB9 1AS Aberdeen, Scotland, United Kingdom.

¶

These authors have contributed equally to this work.

**

Present address: Dept. of Biology, Koshin University, Pusan, 606-080, Korea.

§§

To whom correspondence should be addressed: University of Chicago, MGCB, 920 E. 58th St., Chicago, IL 60637. Tel.: 312-702-1331; Fax: 312-702-3172; wepstein{at}midway.uchicago.edu.

()

The abbreviation used is: kb, kilobase(s).

()

A. Siebers, K. Altendorf, and W. Epstein, unpublished data.

()

A. Treuner, and P. Duerre, personal communication.

ACKNOWLEDGEMENTS

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Hamamoto, J. Marui, K. Matsuoka, K. Higashi, K. Igarashi, T. Nakagawa, T. Kuroda, Y. Mori, Y. Murata, Y. Nakanishi, et al. Characterization of a Tobacco TPK-type K+ Channel as a Novel Tonoplast K+ Channel Using Yeast Tonoplasts J. Biol. Chem., January 25, 2008; 283(4): 1911 - 1920. [Abstract] [Full Text] [PDF]

				C. Davies, R. Shin, W. Liu, M. R. Thomas, and D. P. Schachtman Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation J. Exp. Bot., September 1, 2006; 57(12): 3209 - 3216. [Abstract] [Full Text] [PDF]

				N. Tholema, M. V. d. Bruggen, P. Maser, T. Nakamura, J. I. Schroeder, H. Kobayashi, N. Uozumi, and E. P. Bakker All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus J. Biol. Chem., December 16, 2005; 280(50): 41146 - 41154. [Abstract] [Full Text] [PDF]

				N. Matsuda, H. Kobayashi, H. Katoh, T. Ogawa, L. Futatsugi, T. Nakamura, E. P. Bakker, and N. Uozumi Na+-dependent K+ Uptake Ktr System from the Cyanobacterium Synechocystis sp. PCC 6803 and Its Role in the Early Phases of Cell Adaptation to Hyperosmotic Shock J. Biol. Chem., December 24, 2004; 279(52): 54952 - 54962. [Abstract] [Full Text] [PDF]

				J. Bertrand, K. Altendorf, and M. Bramkamp Amino Acid Substitutions in Putative Selectivity Filter Regions III and IV in KdpA Alter Ion Selectivity of the KdpFABC Complex from Escherichia coli J. Bacteriol., August 15, 2004; 186(16): 5519 - 5522. [Abstract] [Full Text] [PDF]

				E. T. Buurman, D. McLaggan, J. Naprstek, and W. Epstein Multiple Paths for Nonphysiological Transport of K+ in Escherichia coli J. Bacteriol., July 1, 2004; 186(13): 4238 - 4245. [Abstract] [Full Text] [PDF]

				S. J. Ahn, R. Shin, and D. P. Schachtman Expression of KT/KUP Genes in Arabidopsis and the Role of Root Hairs in K+ Uptake Plant Physiology, March 1, 2004; 134(3): 1135 - 1145. [Abstract] [Full Text] [PDF]

				M. van der Laan, M. Gassel, and K. Altendorf Characterization of Amino Acid Substitutions in KdpA, the K+-Binding and -Translocating Subunit of the KdpFABC Complex of Escherichia coli J. Bacteriol., October 1, 2002; 184(19): 5491 - 5494. [Abstract] [Full Text] [PDF]

				T. Nakamura, Y. Fujisaki, H. Enomoto, Y. Nakayama, T. Takabe, N. Yamaguchi, and N. Uozumi Residue Aspartate-147 from the Third Transmembrane Region of Na+/H+ Antiporter NhaB of Vibrio alginolyticus Plays a Role in Its Activity J. Bacteriol., October 1, 2001; 183(19): 5762 - 5767. [Abstract] [Full Text] [PDF]

				N. Uozumi Escherichia coli as an expression system for K+ transport systems from plants Am J Physiol Cell Physiol, September 1, 2001; 281(3): C733 - C739. [Abstract] [Full Text] [PDF]

				A. A. Sardesai and J. Gowrishankar Improvement in K+-Limited Growth Rate Associated with Expression of the N-Terminal Fragment of One Subunit (KdpA) of the Multisubunit Kdp Transporter in Escherichia coli J. Bacteriol., June 1, 2001; 183(11): 3515 - 3520. [Abstract] [Full Text]

				Y. Kato, M. Sakaguchi, Y. Mori, K. Saito, T. Nakamura, E. P. Bakker, Y. Sato, S. Goshima, and N. Uozumi Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters PNAS, May 3, 2001; (2001) 101556598. [Abstract] [Full Text]

				N. Uozumi, E. J. Kim, F. Rubio, T. Yamaguchi, S. Muto, A. Tsuboi, E. P. Bakker, T. Nakamura, and J. I. Schroeder The Arabidopsis HKT1 Gene Homolog Mediates Inward Na+ Currents in Xenopus laevis Oocytes and Na+ Uptake in Saccharomyces cerevisiae Plant Physiology, April 1, 2000; 122(4): 1249 - 1260. [Abstract] [Full Text]

				M. Ga{beta}el, T. Mollenkamp, W. Puppe, and K. Altendorf The KdpF Subunit Is Part of the K+-translocating Kdp Complex of Escherichia coli and Is Responsible for Stabilization of the Complex in Vitro J. Biol. Chem., December 31, 1999; 274(53): 37901 - 37907. [Abstract] [Full Text] [PDF]

				F. Rubio, M. Schwarz, W. Gassmann, and J. I. Schroeder Genetic Selection of Mutations in the High Affinity K+ Transporter HKT1 That Define Functions of a Loop Site for Reduced Na+ Permeability and Increased Na+ Tolerance J. Biol. Chem., March 12, 1999; 274(11): 6839 - 6847. [Abstract] [Full Text] [PDF]

				J. M. Wood Osmosensing by Bacteria: Signals and Membrane-Based Sensors Microbiol. Mol. Biol. Rev., March 1, 1999; 63(1): 230 - 262. [Abstract] [Full Text] [PDF]

				N. Uozumi, T. Nakamura, J. I. Schroeder, and S. Muto Determination of transmembrane topology of an inward-rectifying potassium channel from Arabidopsis thaliana based on functional expression in Escherichia coli PNAS, August 18, 1998; 95(17): 9773 - 9778. [Abstract] [Full Text] [PDF]

				E. J. Kim, J. M. Kwak, N. Uozumi, and J. I. Schroeder AtKUP1: An Arabidopsis Gene Encoding High-Affinity Potassium Transport Activity PLANT CELL, January 1, 1998; 10(1): 51 - 62. [Abstract] [Full Text]

				S. Dorus, H. Mimura, and W. Epstein Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis J. Biol. Chem., March 23, 2001; 276(13): 9590 - 9598. [Abstract] [Full Text] [PDF]

				Y. Kato, M. Sakaguchi, Y. Mori, K. Saito, T. Nakamura, E. P. Bakker, Y. Sato, S. Goshima, and N. Uozumi Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters PNAS, May 22, 2001; 98(11): 6488 - 6493. [Abstract] [Full Text] [PDF]