We are interested in the molecular mechanisms by which proteins are transported across or are inserted into the endoplasmic reticulum (ER) membrane. We have dissected both cotranslational protein translocation in mammals and posttranslational translocation in yeast using a combination of biochemical approaches. Both processes could be reproduced with proteoliposomes reconstituted exclusively from phospholipids and highly purified membrane proteins. For cotranslational translocation, only three components are essential: the SRP receptor, the heterotrimeric Sec61p complex, and the "translocating chain-associating membrane (TRAM)" protein. Posttranslational translocation requires a seven component complex, the Sec complex, consisting of the Sec61p complex and the tetrameric Sec62/63p complex. The Sec61p complex is of particular importance. It is the major constituent of a protein-conducting channel, recognizes the signal sequence of a translocation substrate, and binds the ribosome.
Our present work with the mammalian cotranslational system concentrates on the elucidation of the molecular mechanism of the translocation process, and on the structure of ribosome-Sec61p channel complexes (collaboration with the group of Christopher Akey at Boston University). Using crosslinking methods, we also address the problem of how the orientation of membrane proteins is achieved and how they leave the translocation site to become embedded into the phospholipid bilayer.
The current work with the posttranslational system in yeast concerns the mechanism by which the signal sequence is recognized, by which proteins are transported across the membrane, and by which the channel is opened and closed. We have shown that the signal sequence is recognized by intercalation into trans-membrane segments of Sec61p and that Kar2p acts as a molecular ratchet to move the polypeptide through the channel.
We have recently started to work on the homologous translocation system in E. coli. Our goal is to obtain a high-resolution structure of the translocation channel and to elucidate the mechanism by which the ATPase SecA provides the driving force for translocation.
Selected Publications:
Matlack, K.E.S., Plath, K., Misselwitz, B., and Rapoport, T.A. (1997).
Protein transport by purified yeast Sec complex and Kar2p without membranes.
Science 277, 938-941.
Plath, K., Mothes, W., Wilkinson, B.M., Stirling, C.J., and Rapoport, T.A., (1998). Signal sequence recognition in posttranslational protein transport across the yeast ER membrane. Cell 94, 795-807.
Matlack, K.E.S., Mothes, W., and Rapoport, T.A. (1998). Protein translocation:
tunnel vision. Cell 92:381-390.