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Molecular speleology

How proteins squirm out of ribosomes and worm into membranes

Munich, 10/30/2009

Cell membranes are made of a double layer of lipid molecules that is essentially impermeable to proteins. Yet many proteins reside in membranes, while others are secreted through them. The way out of this conundrum lies in channels that open “on demand” to allow secreted proteins to pass through, while diverting others laterally to become intrinsic membrane proteins. In two papers in today’s issue of Science, Professor Roland Beckmann of LMU Munich’s Gene Center and the Department of Chemistry and Biochemistry and his collaborators reveal new details of the architecture of these passageways and the role of the ribosome as a delivery vehicle.

In higher organisms, including yeast, proteins destined for secretion are distinguished by a sequence that directs them to transmembrane pores in the endoplasmic reticulum (ER). The ER is an intracellular network of membranous tubes that serves as a marshalling yard for newly synthesized proteins and distributes them to their final destinations. Proteins can be introduced into the pores either during (cotranslational translocation) or after (post-translational translocation) synthesis by the ribosome. In the first case, the growing protein chain is fed directly from the ribosome through the protein-conducting channel (PCC) of the pore into the lumen of the ER.

Isolated pores consist of three distinct proteins. X-ray crystallography of pores from bacteria revealed that they form an hourglass shape: two funnels separated by a constriction in the middle, with a plug in the outer funnel. Binding of the signal sequence is thought to open the constriction and displace the plug, allowing secretory proteins to traverse the pore. The channel can also open laterally, giving membrane proteins access to the lipid phase.

Professor Beckmann’s group studied a pore complex from yeast that is actually engaged in cotranslational translocation. They succeeded in visualizing not just the pore itself (consisting of the subunits Ssh1-Sbh2-Sss1), but also the attached ribosome bound to a nascent membrane protein. They accomplished this technical tour-de-force by flash-freezing whole complexes in a thin layer of aqueous detergent, and examining the frozen specimens by electron microscopy (cryo-EM). The pattern of electron density reveals the distribution of protein. Image analysis of many individual particles, randomly oriented in the ice layer, permits construction of an “average” three-dimensional model of the complex at high resolution.

Analysis of the resulting model structures revealed that, as expected, the pore complex binds to the exit site of the ribosome, where the nascent protein emerges. Moreover, in the active structure, the nascent peptide could be followed from its growing end through the tunnel and into the PCC. A similar model of the corresponding Sec61 complex from mammals led the team to conclude that, when the signal sequence of their test (membrane) protein is present in the PCC, the lateral gate that provides access to the lipid phase remains closed.

Not only the PCC, but also the ribosome exit tunnel can be difficult to get through. Some proteins emerge from the tunnel in fits and starts, and bacteria, in which mRNA transcription and translation occur concurrently, exploit this phenomenon for regulatory purposes. In the second paper in Science, Beckmann and collaborators -- including Thomas Steitz of Yale University, who shared this year’s Nobel Prize for Chemistry, and Daniel Wilson, Leader of an Independent Junior Research Group at LMU Munich’s Gene Center -- use a cryo-EM model of a stalled bacterial ribosome to probe alternative conformations of the peptide TnaC in the tunnel. TnaC is encoded by a short sequence upstream of tnaA, the gene for the enzyme tryptophanase. When trytophan levels are low (and the enzyme is not needed), the full-length TnaC is translated and the ribosome is released from the mRNA, uncovering a signal in the that prevents transcription of the adjacent tnaA gene by RNA polymerase. The researchers show in unprecedented detail how, when levels of tryptophan are high (and the cell can obtain energy from its degradation), the conformation of the nascent TnaC is altered so that it interacts with the wall of the tunnel, effectively blocking the exit. These interactions in turn inhibit protein synthesis at the peptidyltransferase center of the ribosome and translation stalls. The result is that TnaC is not released, the signal that would inhibit transcription of tnaA remains concealed by the ribosome, and the downstream genes can be expressed. So, like spelunkers exploring their favourite haunts, proteins sometimes get stuck in tunnels and need help getting out (in this case, a diet low in tryptophan).  (PH)

Publication:
“Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome”
Thomas Becker, Shashi Bhushan, Alexander Jarasch, Jean-Paul Armache, Soledad Funes, Fabrice Jossinet, James Gumbart, Thorsten Mielke, Otto Berninghausen, Klaus Schulten, Eric Westhof, Reid Gilmore, Elisabet Mandon and Roland Beckmann
Science, 29 October 2009

“Structural insight into nascent polypeptide chain-mediated translational stalling”
Birgit Seidelt, C. Axel Innis, Daniel L. Wilson, Marco Gartmann, Jean-Paul Armache, Elizabeth Villa, Leonardo G. Trabuco, Thomas Becker, Thorsten Mielke, Klaus Schulten, Thomas A. Steitz and Roland Beckmann
Science, 29 October 2009

Contact:
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
e-Mail: beckmann@lmb.uni-muenchen.de

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