This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yagur-Kroll, S.
Right arrow Articles by Amster-Choder, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yagur-Kroll, S.
Right arrow Articles by Amster-Choder, O.

 Previous Article  |  Next Article 

Journal of Bacteriology, May 2009, p. 3086-3094, Vol. 191, No. 9
0021-9193/09/$08.00+0     doi:10.1128/JB.01037-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Spatial Arrangement of the β-Glucoside Transporter from Escherichia coli{triangledown} ,{dagger}

Sharon Yagur-Kroll, Ayelet Ido, and Orna Amster-Choder*

Department of Molecular Biology The Hebrew University—Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel

Received 27 July 2008/ Accepted 18 February 2009

The Escherichia coli BglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of β-glucosides across the cytoplasmic membrane. Despite intensive studies of PTS permeases, the mechanism that couples sugar translocation to phosphorylation and the nature of the translocation apparatus are poorly understood. Like many PTS permeases, BglF consists of a transmembrane domain, which in addition to transmembrane helices (TMs) contains a big cytoplasmic loop and two hydrophilic domains, one containing a conserved cysteine that phosphorylates the incoming sugar. We previously reported that the big hydrophilic loop, which connects TM VI to TM VII, contains regions that alternate between facing-in and facing-out states and speculated that it is involved in creating the sugar translocation channel. In the current study we used [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), a membrane-impermeative thiol-specific reagent, to identify sites that are involved in sugar transport. These sites map to the regions that border the big loop. Using cross-linking reagents that penetrate the cell, we could demonstrate spatial proximity between positions at the center of the big loop and the phosphorylation site, suggesting that the two regions come together to execute sugar phosphotransfer. Additionally, positions on opposite ends of the big loop were found to be spatially close. Cys accessibility analyses suggested that the sugar induces a change in this region. Taken together, our results demonstrate that the big loop participates in creating the sugar pathway and explain the observed coupling between translocation of PTS sugars from the periplasm to the cytoplasm and their phosphorylation.

* Corresponding author. Mailing address: Department of Molecular Biology The Hebrew University—Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel. Phone: 972 2 675 8460. Fax: 972 2 6747910. E-mail: amster{at}cc.huji.ac.il

{triangledown} Published ahead of print on 27 February 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, May 2009, p. 3086-3094, Vol. 191, No. 9
0021-9193/09/$08.00+0     doi:10.1128/JB.01037-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»