This project has two primary aims.
The first aim i s to understand how proteins that are destined to travel through the secretory pathway are targeted to transport sites in the endoplasmic reticulum (ER) or the bacterial inner membrane (IM). For the last decade we have been investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its membrane-bound receptor in this process. Although SRP was initially believed to exist only in eukaryotic cells, the sequencing of a large number of microbial genomes has demonstrated that the particle is found in most (if not all) organisms. Previous studies have shown that in mammalian cells SRP recognizes the """"""""signal sequences"""""""" found on virtually all secreted and membrane proteins as they emerge during translation and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. A few years ago we demonstrated that bacterial SRP has a somewhat more restricted function in that it only targets integral membrane proteins to the IM. Consistent with the work of other laboratories, we found that most secreted proteins, by contrast, are targeted to the IM by molecular chaperones. In recent studies we have continued to analyze protein targeting mechanisms and protein targeting signals in bacteria. In the past year we have focused on the targeting of a class of very large (~100-400 kD) secreted toxins called autotransporters. These proteins are produced by a wide range of pathogenic Gram negative bacteria and often contain exceptionally long signal peptides that are distinguished by a unique N-terminal sequence motif. Although work by others has suggested that the unusual signal peptides are recognized by SRP, we have found that a model autotransporter produced by E. coli O157:H7 called EspP is targeted to the IM by an SRP-independent pathway. Surprisingly, we found that the unusual signal peptide was not required for translocation of EspP across the IM, but instead was essential for late stages of protein biogenesis that occur after the protein is translocated across the IM. These results not only provide insight into the biogenesis of a class of proteins associated with bacterial pathogenesis, but also provide evidence for a novel signal peptide function.
The second aim of the project is to elucidate the function of factors that facilitate the transport of proteins across or insertion into the ER or bacterial IM. In the last year we have continued to study the structure and membrane distribution of YidC, a highly conserved bacterial protein that has been shown to play an important but still poorly defined role in membrane protein biogenesis. Using a variety of different biochemical methods we have obtained evidence that YidC exists in a homooligomeric form similar to that observed for its mitochondrial homolog (a protein called Oxa1p). The results suggest that YidC is a multisubunit complex that could function as a protein conducting channel. In addition, recent results indicate that YidC is freely mobile within the IM and call into question results published by another group suggesting that YidC has a strictly polar localization.
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