Secretory proteins, most integral membrane proteins and the content proteins of all organelles in the exocytic and endocytic membrane systems are initially translocated across or integrated into the rough endoplasmic reticulum (RER). The efficiency and fidelity of the protein translocation and membrane protein integration pathways are essential for viability. The core machinery of the protein translocation pathways is evolutionarily conserved in all organisms. The objective of this research proposal is to address important questions concerning the structure and function of eukaryotic protein translocation channels using the yeast Saccharomyces cerevisiae as our primary experimental system. Protein translocation across the yeast RER occurs by cotranslational and posttranslational translocation pathways. Sec61p is the core subunit of a heterotrimeric cotranslational translocation channel. The Sec61 heterotrimer assembles with Sec62/Sec63 complex to form the heptameric SEC complex that has a well-described role in posttranslational translocation of proteins across the RER. The objective of the first specific aim is to analyze the integration of membrane proteins that have the ER-targeting signal located in the second half of the protein. Proteins with late internal targeting signals are a conserved class of membrane protein that has not been carefully investigated to analyze the mechanism of targeting and integration. The objective of the second specific aim is test the hypothesis that the Sec62/Sec63 complex has a role in the cotranslational translocation of secreted proteins and integration of multi-spanning membrane proteins. Multiple approaches will be used to inactivate the Sec62/Sec63 complex. We will explicitly test whether mutations in the Sec62/Sec63 complex cause defects in translocation and membrane protein integration by a direct or indirect mechanism. We will test the hypothesis that ribosome-nascent chain (RNC) complexes are targeted to the SEC complex by a previously unidentified mechanism. The third specific aim of the project is to obtain a structure of a native mammalian translocon consisting of the Sec61 complex, the TRAP complex and the oligosaccharyltransferase by cryoelectron microscopy. A second objective is to obtain a structure of a yeast RNC-SEC complex by cryoelectron microsocopy.
The biosynthesis of secretory proteins, lysosomal proteins and integral membrane proteins is of fundamental importance to human health. Mutations in the human SEC63 gene cause autosomal dominant polycystic liver and kidney disease due to reductions in the biosynthesis of polycystin-1. Homozygous SEC63 gene knockouts cause an early embryonic lethal phenotype in mice. Defects in the biosynthesis, modification and folding of proteins in the rough endoplasmic reticulum are responsible for a growing number of pathologies that are collectively referred to as 'ER quality control' diseases. Our studies, which are directed towards understanding the molecular mechanism of protein translocation and membrane protein integration, will provide insight into these crucial events in protein biosynthesis.
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