PROJECT SUMMARY The identities and functions of cellular membrane-bound compartments such as the endoplasmic reticulum (ER), mitochondria, and synaptic vesicles, are largely determined by protein composition. Organelle dysfunction and impaired membrane protein quality control (QC) are hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's, Parkinson's, and Huntington's disease. Molecular- level insights into the mechanisms that ensure high-fidelity membrane protein biogenesis are required to understand how neurodegenerative diseases develop and identify effective treatments. All membrane proteins face two fundamental biosynthetic challenges. First, they must localize to the correct cellular membrane. Second, they must insert hydrophobic transmembrane domains into target membranes in the correct orientation. It is not known how cells meet these basic biosynthetic requirements for the diverse membrane proteins that make up 25-30% of the proteome. Using single-spanning membrane proteins as models, we have established experimental systems of membrane protein biosynthesis and QC that are tractable for mechanistic dissection. With these systems, we recently discovered that the ER-resident transporter ATP13A1 dislocates mislocalized mitochondrial membrane proteins. Protein dislocation by ATP13A1 provides opportunities for correct targeting and is required to maintain mitochondrial protein localization. In this proposal, we will leverage ATP13A1 function as a molecular handle to study the mechanisms that lead to, recognize, and eliminate aberrant proteins at the ER. In Aim 1, we will identify the biosynthetic factors that aberrantly insert mitochondrial membrane proteins into the ER and determine how these mechanisms contribute to mitochondrial protein homeostasis. In Aim 2, we will investigate the QC mechanisms that selectively recognize and target mislocalized mitochondrial membrane proteins for ER-associated degradation (ERAD). In Aim 3, we will investigate the topogenesis of type II membrane proteins that should insert into the ER with their N- terminus in the cytosol. Because a subset of type II proteins is selectively destabilized by ATP13A1 depletion, we hypothesize that these proteins harbor specific features prone to insertion in the wrong orientation, resulting in the need for ATP13A1-mediated dislocation. Completion of this project will reveal mechanisms that mis- insert membrane proteins into the ER, generate a mechanistic model of a mammalian ERAD pathway, and shed light on how a handful of biosynthetic and QC factors handle a large and diverse clientele. Altogether, these findings will reveal molecular-level insights into membrane protein QC target selection and mechanistic principles underlying how cellular biosynthetic and QC mechanisms collaborate to ensure the integrity of membrane protein biogenesis needed to preserve neuronal function.

PROJECT NARRATIVE Organelle dysfunction and compromised membrane protein quality control are hallmarks of neurodegenerative disorders. This proposal investigates the molecular mechanisms that collaborate to ensure high-fidelity membrane protein localization and topology. Molecular-level insights into these fundamental protein biosynthetic and quality control processes are necessary to understand how neurodegenerative diseases develop and identify effective treatments.