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Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 31, 26052-26059 p.Article in journal (Refereed) Published
Abstract [en]

The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.

Place, publisher, year, edition, pages
2012. Vol. 287, no 31, 26052-26059 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-80590DOI: 10.1074/jbc.M112.357590ISI: 000306916300032OAI: oai:DiVA.org:su-80590DiVA: diva2:557708
Note

AuthorCount:6;

Available from: 2012-09-28 Created: 2012-09-25 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Dual-topology membrane proteins in Escherichia coli
Open this publication in new window or tab >>Dual-topology membrane proteins in Escherichia coli
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular life, as we know it, is absolutely dependent on biological membranes; remarkable superstructures made of lipids and proteins. For example, all living cells are surrounded by at least one membrane that protects the cell and holds it together. The proteins that are embedded in the membranes carry out a wide variety of key functions, from nutrient uptake and waste disposal to cellular respiration and communication. In order to function accurately, any integral membrane protein needs to be inserted into the cellular membrane where it belongs, and in that particular membrane it has to attain its proper structure and find partners that might be required for proper function. All membrane proteins have evolved to be inserted in a specific overall orientation, so that e.g. substrate-binding parts are exhibited on the ‘right side’ of the membrane. So, what determines in which way a membrane protein is inserted? Are all membrane proteins inserted just so?

The focus of this thesis is on these fundamental questions: how, and when, is the overall orientation of a membrane protein established? A closer look at the inner membrane proteome of the familiar gram-negative bacterium Escherichia coli revealed a small group of proteins that, oddly enough, seemed to be able to insert into the membrane in two opposite orientations. We could show that these dual-topology membrane proteins are delicately balanced, and that even the slightest manipulations make them adopt a fixed orientation in the membrane. Further, we show that these proteins are topologically malleable until the very last residue has been synthesized, implying interesting questions about the topogenesis of membrane proteins in general. In addition, by looking at the distribution of homologous proteins in other organisms, we got some ideas about how membrane proteins might evolve in size and complexity. Structural data has revealed that many membrane bound transporters have internal, inverted symmetries, and we propose that perhaps some of these proteins derive from dual-topology ancestors.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 66 p.
Keyword
membrane protein topology, dual-topology, evolution, Escherichia coli
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-61944 (URN)978-91-7447-351-3 (ISBN)
Public defence
2011-10-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2011-10-06 Created: 2011-09-05 Last updated: 2013-04-22Bibliographically approved
2. EmrE, a puzzling transporter: Assembly, biogenesis and evolution of a dual-topology membrane protein
Open this publication in new window or tab >>EmrE, a puzzling transporter: Assembly, biogenesis and evolution of a dual-topology membrane protein
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological membranes are the key to cell existence, as they are able to both isolate and connect their interior with the environment. Membranes are composed of lipids and proteins that create a semi-permeable barrier; because the lipid bilayer stops free diffusion of most molecules and ions, membrane proteins play an important role in connecting the interior of the cell with its environment. They function as receptors, sensing signals to trigger a response; cell adhesion molecules, holding neighboring cells together, or transporters and channels importing nutrients and extruding waste, among other chemical compounds, in a controlled manner.

In order for membrane proteins to function correctly, proper insertion, folding and oligomerization in the bilayer is essential. While most membrane proteins adopt a unique orientation in the membrane, some proteins adopt multiple topologies. A well-known case is the dual-topology membrane proteins that adopt two opposite orientations in the membrane. The best-studied dual-topology protein is EmrE, a dimeric multidrug transporter found in Escherichia coli, and other bacteria.

The existence of dual-topology proteins raises many questions regarding oligomerization, biogenesis and evolution of membrane proteins. In this thesis, EmrE has been used as a model protein to study some of these issues. Our goals were (i) to settle the controversy regarding whether the arrangement of the monomers within the EmrE dimer is parallel or antiparallel, (ii) to test the validity of the published X-ray structure by in vivo experiments and, (iii) to elucidate the mechanism of membrane insertion (iv) and the evolution of dual-topology membrane proteins.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 68 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-94830 (URN)978-91-7447-797-9 (ISBN)
Public defence
2013-12-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Epub ahead of print; Paper 4: Manuscript

Available from: 2013-11-14 Created: 2013-10-15 Last updated: 2013-11-05Bibliographically approved

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