This project has two primary aims.
The first aim i s to understand how proteins which are secreted or inserted into membranes are targeted to transport sites in the endoplasmic reticulum (ER) or, equivalently, the bacterial cytoplasmic membrane. In particular we are investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its ER-bound receptor in this process. Previous studies have shown that SRP recognizes nascent polypeptide chains containing """"""""signal sequences"""""""" and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. We hope to elucidate the mechanism by which SRP recognizes the diverse family of signal sequences found on different proteins and then releases them at the surface of the ER in a regulated fashion. By studying this problem we expect to obtain insight into the function and regulation of proteins that possess broad substrate specificity.
The second aim of the project is to elucidate the mechanism by which the highly conserved transport channel or """"""""translocon"""""""" (called the Sec61p complex in eukaryotes and the SecY complex in bacteria) facilitates both protein translocation and membrane protein insertion, two seemingly distinct processes. We expect that these experiments will provide significant insight into the function of membrane-bound channels. In recent work we have focused on the function of E. coli homologs of SRP, the SRP receptor, and SecY using a combination of biochemical and genetic methods. The function of SRP in bacteria has been of particular interest in light of substantial evidence indicating that unlike mammalian cells, bacteria utilize predominantly SRP-independent mechanisms to target secreted proteins to the cytoplasmic membrane. We have found that E. coli SRP, like its eukaryotic counterpart, interacts with nascent polypeptides at a specific step of the ribosome cycle. The bacterial particle, however, targets only inner membrane proteins (IMPs) to the cytoplasmic membrane, and therefore has a much more restricted function than its mammalian counterpart. Interestingly, we have found that a subset of IMPs do not appear to require SRP for their insertion into the membrane. By analyzing these proteins, we are beginning to identify the molecular determinants of SRP-dependence. In addition, we have obtained strong evidence that the SecA protein, a peripheral membrane protein that has previously been shown to be required for the post-translational insertion of secreted proteins, is also required for the insertion of IMPs that are targeted by the SRP pathway. This finding was unexpected in light of previous studies on the SRP pathway in mammalian cells and suggests that some aspects of the protein translocation process differ in eukaryotes and prokaryotes. Finally, we have identified a secY mutation (secY40) that has distinct effects on IMP insertion and protein export. Our studies on this allele provide the first evidence that the protein translocation and membrane protein insertion functions of the translocon can be separated genetically.
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