9722889 McCracken Eukaryotic cells have an intracellular protein degradation process that removes aberrant and unassembled proteins from the endoplasmic reticulum (ER). This ER-Associated protein Degradation pathway (ERAD) is highly selective for specific soluble and integral membrane proteins, while the majority or ER resident and secreted proteins are quite stable. Evidence that the ER chaperone calnexin has a role in ERAD indicates that molecular chaperones are required for this remarkable substrate selectivity, yet the mechanism of chaperone action in this context is unclear. Furthermore, although previous studies suggested that ERAD was a novel intracellular proteolytic process involving unidentified ER- localized proteases, a recent and unexpected finding has made it clear that the quality control of proteins in the ER includes the export of aberrant and unassembled proteins to the cytosol for degradation. The mechanism of retrograde protein transport across the ER membrane, and the role of molecular chaperones in this process, are the major questions addressed by the research proposed here. The specific aims are to: 1, determine which molecular chaperones are required for ERAD and investigate the mechanism of substrate selectivity and targeting; 2, verify whether the protein translocation channel, Sec61p, known to be the route through which nascent proteins enter the ER, is indeed also responsible for the retrograde translocation of a soluble yeast ERAD substrate; and 3, to identify new proteins involved in ERAD through molecular cloning of the wild-type alleles of ERAD-defective genes. These studies will be facilitated by a cell free system that has been developed that allows the dissection of ERAD at the molecular level, and by ERAD-defective yeast mutants that have already been isolated. Together, these advances provide a unique opportunity to combine biochemical and genetic approaches to elucidate the components and the molecular mechanisms a nd regulation of ERAD. The first two aims will be accomplished using the reconstituted in vitro assay and chemical crosslinking to reveal direct and temporal associations between the ERAD substrate, the molecular chaperones, and a protein component of the translocation complex. It is anticipated that these studies will raise new questions regarding the identity an role of yet-unidentified factors involved in ERAD. To accomplish the third aim, the mutants will each by studied by the in vitro assay to determine whether a given mutant strain is defective in substrate selectivity, transport of ERAD substrate to the cytosol, targeting of substrate to the cytosolic proteasome, or proteolysis. In addition, the wild type alleles of the defective ERAD genes will be cloned to identify new proteins involved in ERAD. Cells have quality-control processes in place to ensure that newly-synthesized proteins are structurally correct so that they will function properly. Many proteins are synthesized for either export (secretion) or use in specialized intracellular compartments such as the lysosome; such proteins are sequestered early in their synthesis into a subcellular compartment known as the endoplasmic reticulum (ER). The quality control system within the ER which is responsible for degradation of ER-associated aberrant proteins has been given the acronym "ERAD." It has long been assumed that when proteins in the ER are recognized as aberrant, the degradation of those proteins takes place within the ER. Drs. McCracken and Brodsky, the Principal and co-Principal Investigators of this project, have recently made the unexpected discovery that proteins recognized as aberrant in the ER are actually translocated back into the cytosol (possibly through the same pore in the membrane through which they were brought in originally) and degraded via proteolytic machinery in the cytosol known as proteasomes. The combined biochemical and molecular genetic approach proposed in t his project will lead to an understanding of how this process actually works. ***
