HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 44, 42942-42949, October 31, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/44/42942    most recent M308253200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pirch, T. Articles by Jung, H. Search for Related Content PubMed PubMed Citation Articles by Pirch, T. Articles by Jung, H. Social Bookmarking                      What's this?

Transmembrane Domain II of the Na⁺/Proline Transporter PutP of Escherichia coli Forms Part of a Conformationally Flexible, Cytoplasmic Exposed Aqueous Cavity within the Membrane^*

Torsten Pirch, Silke Landmeier, and Heinrich Jung

From the Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany

The Na⁺/proline transporter PutP of Escherichia coli is a member of a large family of Na⁺/substrate symporters. Previous work on PutP suggests an involvement of the region ranging from Asp-55 to Gly-58 in binding of Na⁺ and/or proline (Pirch, T., Quick, M., Nietschke, M., Langkamp, M., Jung, H. (2002) J. Biol. Chem. 277, 8790–8796). In this study, a complete Cys scanning mutagenesis of transmembrane domain II (TM II) of PutP was performed to further elucidate the role of the TM in the transport process. Strong defects of PutP function were observed upon substitution of Ala-48, Ala-53, Trp-59, and Gly-63 by Cys in addition to the previously characterized residues Asp-55, Ser-57, and Gly-58. However, except for Asp-55 none of these residues proved essential for function. The activity of eight mutants was sensitive to N-ethylmaleimide inhibition with the sensitive positions clustering predominantly on a hydrophilic face in the cytoplasmic half of TM II. The same face was also highly accessible to the bulky sulfhydryl reagent fluorescein 5-maleimide in randomly oriented membrane vesicles, suggesting an unrestricted accessibility of the corresponding amino acid positions via an aqueous pathway. Na⁺ stimulated the reactivity of Cys toward fluorescein 5-maleimide at two positions while proline inhibited reaction of the sulfhydryl group at nine positions. Taken together, the results demonstrate that TM II of PutP is of particular functional importance. It is proposed that hydrophilic residues in the cytoplasmic half of TM II participate in the formation of an aqueous cavity in the membrane that allows Na⁺ and/or proline binding to residues located in the middle of the TM (e.g. Asp-55 and Ser-57). In addition, the data indicate that TM II participates in Na⁺- and proline-induced conformational alterations.

Received for publication, July 29, 2003

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 431-P10. 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. Fax: 49-541-9692870; E-mail: jung_h{at}biologie.uni-osnabrueck.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Yagur-Kroll, A. Ido, and O. Amster-Choder Spatial Arrangement of the {beta}-Glucoside Transporter from Escherichia coli J. Bacteriol., May 1, 2009; 191(9): 3086 - 3094. [Abstract] [Full Text] [PDF]

				K. Nanatani, P. C. Maloney, and K. Abe Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT J. Bacteriol., April 1, 2009; 191(7): 2122 - 2132. [Abstract] [Full Text] [PDF]

				S. Faham, A. Watanabe, G. M. Besserer, D. Cascio, A. Specht, B. A. Hirayama, E. M. Wright, and J. Abramson The Crystal Structure of a Sodium Galactose Transporter Reveals Mechanistic Insights into Na+/Sugar Symport Science, August 8, 2008; 321(5890): 810 - 814. [Abstract] [Full Text] [PDF]

				A. Tolia, K. Horre, and B. De Strooper Transmembrane Domain 9 of Presenilin Determines the Dynamic Conformation of the Catalytic Site of {gamma}-Secretase J. Biol. Chem., July 11, 2008; 283(28): 19793 - 19803. [Abstract] [Full Text] [PDF]

				G. Christie, M. Lazarevska, and C. R. Lowe Functional Consequences of Amino Acid Substitutions to GerVB, a Component of the Bacillus megaterium Spore Germinant Receptor J. Bacteriol., March 15, 2008; 190(6): 2014 - 2022. [Abstract] [Full Text] [PDF]

				D. Hilger, M. Bohm, A. Hackmann, and H. Jung Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli in Ligand Binding and Transport J. Biol. Chem., February 22, 2008; 283(8): 4921 - 4929. [Abstract] [Full Text] [PDF]

				T. Mascher, J. D. Helmann, and G. Unden Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 910 - 938. [Abstract] [Full Text] [PDF]

				Z. Zhang, Y.-Y. Liu, and S. M. Jhiang Cell Surface Targeting Accounts for the Difference in Iodide Uptake Activity between Human Na+/I- Symporter and Rat Na+/I- Symporter J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6131 - 6140. [Abstract] [Full Text] [PDF]

				I. Sobczak and J. S. Lolkema Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae J. Biol. Chem., July 23, 2004; 279(30): 31113 - 31120. [Abstract] [Full Text] [PDF]

				W. Zhang, M. Bogdanov, J. Pi, A. J. Pittard, and W. Dowhan Reversible Topological Organization within a Polytopic Membrane Protein Is Governed by a Change in Membrane Phospholipid Composition J. Biol. Chem., December 12, 2003; 278(50): 50128 - 50135. [Abstract] [Full Text] [PDF]