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Dual-topology membrane proteins in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Gunnar von Heijne)
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 [en]
membrane protein topology, dual-topology, evolution, Escherichia coli
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-61944ISBN: 978-91-7447-351-3 (print)OAI: oai:DiVA.org:su-61944DiVA: diva2:443191
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
List of papers
1. Identification and evolution of dual-topology membrane proteins
Open this publication in new window or tab >>Identification and evolution of dual-topology membrane proteins
2006 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 13, no 2, 112-116 p.Article in journal (Refereed) Published
Abstract [en]

Integral membrane proteins are generally believed to have unique membrane topologies. However, it has been suggested that dual-topology proteins that adopt a mixture of two opposite orientations in the membrane may exist. Here we show that the membrane orientations of five dual-topology candidates identified in Escherichia coli are highly sensitive to changes in the distribution of positively charged residues, that genes in families containing dual-topology candidates occur in genomes either as pairs or as singletons and that gene pairs encode two oppositely oriented proteins whereas singletons encode dual-topology candidates. Our results provide strong support for the existence of dual-topology proteins and shed new light on the evolution of membrane-protein topology and structure.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-23012 (URN)10.1038/nsmb1057 (DOI)
Available from: 2006-10-29 Created: 2006-10-29 Last updated: 2017-12-13Bibliographically approved
2. Emulating membrane protein evolution by rational design
Open this publication in new window or tab >>Emulating membrane protein evolution by rational design
2007 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 315, no 5816, 1282-1284 p.Article in journal (Refereed) Published
Abstract [en]

How do integral membrane proteins evolve in size and complexity? Using the small multidrug-resistance protein EmrE from Escherichia coli as a model, we experimentally demonstrated that the evolution of membrane proteins composed of two homologous but oppositely oriented domains can occur in a small number of steps: An original dual-topology protein evolves, through a gene-duplication event, to a heterodimer formed by two oppositely oriented monomers. This simple evolutionary pathway can explain the frequent occurrence of membrane proteins with an internal pseudo–two-fold symmetry axis in the plane of the membrane.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-23013 (URN)10.1126/science.1135406 (DOI)000244564700051 ()
Available from: 2006-10-29 Created: 2006-10-29 Last updated: 2017-12-13Bibliographically approved
3. Control of Membrane Protein Topology by a Single C-Terminal Residue
Open this publication in new window or tab >>Control of Membrane Protein Topology by a Single C-Terminal Residue
Show others...
2010 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 328, no 5986, 1698-1700 p.Article in journal (Refereed) Published
Abstract [en]

The mechanism by which multispanning helix-bundle membrane proteins are inserted into their target membrane remains unclear. In both prokaryotic and eukaryotic cells, membrane proteins are inserted cotranslationally into the lipid bilayer. Positively charged residues flanking the transmembrane helices are important topological determinants, but it is not known whether they act strictly locally, affecting only the nearest transmembrane helices, or can act globally, affecting the topology of the entire protein. Here we found that the topology of an Escherichia coli inner membrane protein with four or five transmembrane helices could be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C terminus. This observation points to an unanticipated plasticity in membrane protein insertion mechanisms.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-50108 (URN)10.1126/science.1188950 (DOI)000279107400044 ()
Note

authorCount :5

Available from: 2010-12-29 Created: 2010-12-21 Last updated: 2017-12-11Bibliographically approved
4. Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
Open this publication in new window or tab >>Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers
Show others...
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-80590 (URN)10.1074/jbc.M112.357590 (DOI)000306916300032 ()
Note

AuthorCount:6;

Available from: 2012-09-28 Created: 2012-09-25 Last updated: 2017-12-07Bibliographically approved

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