During the synthesis of secretory and membrane proteins, polypeptides move through a universally conserved protein-conducting channel, formed by the Sec61/SecY complex that is located in the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. The channel operates in two different modes depending on its binding partners. In co-translational translocation, a pathway found in all organisms, the channel associates with a translating ribosome. In post-translational translocation, the channel cooperates with either the Sec62–Sec63 complex in eukaryotes or the SecA ATPase in bacteria. Despite tremendous progress in our understanding of protein translocation over the past decades, many questions about its mechanism remain to be answered. These include (1) how the channel maintains the membrane barrier for small molecules while transporting large proteins, (2) what is the functional implication of channel oligomerization, and (3) how the channel interacts with binding partners and polypeptide substrates during translocation. To address these questions, we developed a novel in vivo method to generate both co- and post-translation translocation intermediates in intact Escherichia coli cells, such that polypeptide chains are only partially translocated through the channel. Using this method, we first demonstrated that a translocating polypeptide itself blocks small molecules from passing through an open SecY channel. A hydrophobic pore ring surrounding the polypeptide chain is vital for maintaining the membrane barrier during translocation. Next, we examined the importance of SecY oligomerization in protein translocation. Crosslinking experiments showed that SecY molecules interact with each other in native membranes, but that this self-association is greatly decreased upon insertion of polypeptide substrates. We also showed that SecY mutants that cannot form oligomers are still functional in vivo. Collectively, our data indicate that a single copy of SecY is sufficient for protein translocation. Finally, we isolated an intact co-translational translocation intermediate from E. coli cells and analyzed its structure by cryo-electron microscopy. An initial map shows a translating ribosome containing all three tRNAs is bound to one copy of the SecY channel. Analysis of a large dataset is ongoing in order to understand the structural basis of how the channel interacts with the ribosome and translocating nascent chain.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10288619 |
| Date | January 2012 |
| Creators | Park, Eunyong |
| Contributors | Rapoport, Tom A. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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