PROJECT SUMMARY The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. One aspect of this proposal is to clarify how misfolded endoplasmic reticulum (ER) proteins are retro-translocated from the ER into the cytosol (ER-associated protein degradation, ERAD). The proposal is based on recently determined cryo-electron microscopy (cryoEM) structures of the Hrd1 complex that elucidated how luminal proteins are retro-translocated, and on experiments that clarified how the Cdc48 ATPase processes its substrates. Another aspect of the proposal deals with protein import into peroxisomes. Our recent results indicate that folded cargo translocates with its receptor into the lumen of peroxisomes; the receptor subsequently returns to the cytosol through a retro-translocon formed by a heterotrimeric ubiquitin ligase complex. Here, we will address central outstanding questions of these protein translocation pathways: Specific aim #1: How are proteins moved from the ER lumen across the membrane? We will clarify the mechanism by which misfolded glycoproteins are delivered to the Hrd1 complex and moved through the membrane. Specific aim #2: How are substrates moved from the ER membrane to the proteasome? We propose to determine how the Cdc48 complex moves substrates into the cytosol and test the hypothesis that membrane proteins are extracted together with bound lipids. We will investigate how shuttling factors and Cdc48- binding cofactors transfer substrates between Cdc48 and the 26S proteasome. Specific aim #3: How are proteins imported into peroxisomes? Based on preliminary experiments, we propose to test the hypothesis that Pex13 forms a nuclear pore-like structure through which cargo-bound receptors move across the peroxisomal membrane. Specific aim #4: How are receptors recycled back into the cytosol? We will test our model that the ubiquitin ligase complex forms a retro-translocation channel for recycling receptors. We will use purified proteins to reconstitute ubiquitination by the ligase complex and the subsequent membrane extraction by the Pex1/Pex6 ATPase. The mechanism of ERAD and of peroxisomal protein import are of great medical importance. Many diseases, including cystic fibrosis, are caused by the misfolding of ER proteins and their degradation. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference. Our work may also lead to a better understanding and treatment of peroxisomal disorders, such as Zellweger syndrome.