Tom A. Rapoport
Department of Cell Biology
tel: (617) 432-0637; fax: (617) 432-1190
We are interested in the molecular mechanisms by which proteins are transported across the eukaryotic endoplasmic reticulum (ER) membrane or across the bacterial plasma membrane. Proteins are transported through a protein-conducting channel, formed from a conserved trimeric membrane protein complex, called the Sec61p complex in eukaryotes, and the SecY complex in prokaryotes. The channel is a passive pore that needs to associate with partners that provide the driving force for translocation. We know of three different pathways in which the channel functions.
The first is co-translational translocation, in which the ribosome is the major partner. Our present work concentrates on the structure of ribosome-channel complexes using electron cryo-microscopy single particle analysis (collaboration with the group of Christopher Akey at Boston University) and X-ray analysis.
The second mode is post-translational translocation in eukaryotes, in which another membrane protein complex, the Sec62/63p complex, and the luminal BiP protein are the partners.
The third mode is posttranslational translocation in bacteria, in which the ATPase SecA is the major partner. Our goal is to elucidate the mechanism by which the ATPase SecA provides the driving force for translocation.
A major effort in the lab is directed towards high-resolution structures of the translocation channel. We have obtained X-ray structures of the SecY complex from an archaebacterium, and more recently, of a SecA-channel complex. Together with biochemical data, these results suggest mechanisms for how the signal sequence of a substrate is recognized, how polypeptide chains are moved through the channel, and how trans-membrane segments of membrane proteins are integrated into the lipid bilayer.
We are also interested in the process by which misfolded ER proteins are transported back into the cytosol for degradation by the proteasome, a process called ERAD (for ER-associated protein degradation) or retro-translocation. We have provided evidence that there exist three ERAD pathways in yeast, depending on where the misfolded domain of the ER protein is located (ERAD-L for proteins with misfolded luminal domains, ERAD-M for proteins with misfolded intra-membrane domains, and ERAD-C for membrane proteins with misfolded cytosolic domains). The pathways use different ubiquitin-ligase complexes but converge at the Cdc48p ATPase complex (p97 or VCP complex in mammals). We are currently using translocation intermediates and crosslinking methods to identify the retro-translocation channel that likely exists at least for ERAD-L substrates.
A major effort in the lab concerns the mechanism by which the structure of the ER is generated and maintained. Our results show that the reticulons and DP1/Yop1p shape the tubular ER. We are interested in understanding how these proteins are regulated and how ER sheets are generated.
Other Projects Include:
van den Berg, L., Clemons, W., Collinson, I., Hartmann, E., Modis, Y., Harrison, S.C., and Rapoport, T.A. (2004). X-ray structure of a protein-conducting channel. Nature 427: 36-44.
Carvalho, P., Goder, V., and Rapoport T.A. (2006). Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126: 361-373.
Voeltz, G.K., Prinz, W.A., Shibata, Y., Rist, J.M., and Rapoport, T.A. (2006). A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124: 573-86.
Rapoport, T.A. (2007). Protein translocation across the eukaryotic ER and bacterial plasma membranes. Nature 450: 663-669.
Zimmer, J., Nam, Y., and Rapoport, T.A. (2008). Structure of a complex of the ATPase SecA protein-translocation channel. Nature 455(7215): 936-43.
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