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Crossing a soapy sea –

3D images reveal how proteins are inserted into the cell membrane

Munich, 04/17/2011

All cells are bounded by a so-called plasma membrane and also contain several internal membrane systems. The functions of these systems are determined by the proteins incorporated in them. An international team led by Dr. Jens Frauenfeld and Professor Roland Beckmann, who are based at the Gene Center and the Department of Biochemistry at LMU Munich, and are members of the Center for Integrated Protein Science Munich (CiPSM), has developed a new method that allows one to visualize in detail how proteins are inserted into lipid membranes under conditions that mimic those in the cell. The process begins during protein synthesis by the ribosomes, the cell’s protein factories. Ribosomes that are engaged in the production of a protein destined for secretion or plasma membrane insertion attach to a transport pore that traverses the membrane. As the new work shows, at high resolution and in three-dimensional detail, the protein is fed directly into the pore as it emerges from a lateral tunnel in the ribosome. The new method not only reveals precisely how membrane protein integration occurs, it also has the potential to uncover in molecular detail how inhibitors of protein insertion work, and could therefore contribute to the development and optimization of new therapeutic agents that interfere with the process. (Nature Structural and Molecular Biology online, 17  April 2011)

About one-third of all cellular proteins are either secreted by the cell in which they are synthesized or are incorporated into cell membranes as “integral membrane proteins”.  All cell membranes basically consist of a double layer of fat molecules, which makes them virtually impermeable to electrically charged molecules such as proteins. To integrate proteins into bilayer membranes, the cell utilizes so-called translocons. These are special protein complexes that form channels in membranes, which can be opened on demand to allow the import or export of other proteins through the lipid phase. As Professor Beckmann explains, “Where a given membrane protein is inserted is determined by a special sequence called a signal sequence."

The process of membrane protein integration has so far been something of a black box, as it could not be studied directly in cellular membranes. “That is problematical because the functions of many membrane proteins are dependent on the surrounding lipid bilayer,” says Jens Frauenfeld. Together with an international team of collaborators, he and Beckmann have now developed a method that provides structural insights into membrane protein insertion under natural conditions. The key to their success lies in so-called  nanodiscs -- nanometer-sized sheets of bilayered lipids held together by protein rings.

Into such nanodiscs the researchers inserted a protein complex called SecYEG, which serves as a universal transport pore for the secretion of proteins through the plasma membrane. To each disc they then attached a ribosome that was in the process of synthesizing a protein destined for membrane insertion.  “Using three-dimensional cryo-electron microscopy we were able to visualize individual nanodiscs and the protein pore in the bilayer at high resolution, which itself was a significant achievement,” says Frauenfeld. “Three-dimensional reconstructions of the whole complex then enabled us to capture the secreted protein within the gate as it emerges from the ribosome and is fed into the pore in the membrane.” The team was also able to demonstrate for the first time a direct interaction between the ribosome and the lipids, which facilitates incorporation of the protein into the membrane with minimal expenditure of energy.

The transport of proteins across membranes is an integral part of many biological phenomena, including inflammatory responses and cancer. Many therapeutically useful compounds owe their efficacy to their ability to inhibit transmembrane transport. Agents such as the synthetic peptide cotransin act by blocking the transport pore itself. According to Frauenfeld and Beckmann, the use of nanodiscs for structure determination also has therapeutic potential. Because this approach can provide detailed views of transport processes in the context of a  natural lipid environment, it promises to enhance our understanding of the mechanism of action of inhibitors like cotransin, and to improve their efficacy. The investigators hope that this will also make it easier to find new ways of modulating protein secretion and membrane insertion. (göd)

The study was carried out under the auspices of the Center for Integrated Protein Science Munich (CIPSM), and in the context of DFG Priority Programs SFB 594 (Molecular Machines) and SFB 646 (Regulatory Networks in Gene Expression and Maintenance). Jens Frauenfeld’s contribution was also supported by the receipt of a 1000-Euro Prize for Science and Research, awarded by the ROMIUS Foundation, which is sponsored by the pharmaceutical firm Roche.


"Cryo-EM structure of the ribosome-SecYE complex in the membrane environment"
J. Frauenfeld, J. Gumbart, E.O. van der Sluis, S. Funes, M. Gartmann, B. Beatrix, T. Mielke, O. Berninghausen, T. Becker, K. Schulten, R. Beckmann.
Nature Structural and Molecular Biology, Advance Online Publication, 17 April 2011

Prof. Dr. Roland Beckmann
Gene Center Munich
Center for Integrated Protein Science Munich (CIPSM)
Department of Chemistry and Biochemistry
Phone: +49 (0)89/2180 – 76900

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