PROJECT SUMMARY and ABSTRACT The vast majority of biological functions use membrane-embedded proteins. Controlling the quality and quantity of these membrane proteins is critical for these processes to work normally, and defects in the ability to destroy misfolded proteins or getting rid of membrane proteins that are no longer needed cause a plethora of human diseases. A central mechanism used to specify which proteins undergo degradation is attaching ubiquitin to them. Ubiquitin is a small protein that is extremely well-conserved across the entire kingdom of eukaryotic organisms. When ubiquitin is chemically linked to a membrane protein, it causes that protein to be degradaded by cellular machinery that can recognize ubiquitin. Ubiquitin can also be linked to another ubiquitin, forming polyubiquitin chains. Different polyubiquitin chains are made by changing how one ubiquitin is linked to another, and different ubiquitin chains are used by different degradation machines. One machine sorts membrane proteins to the interior of lysosomes by first sorting them into intralumenal vesicles that accumulate within multivesicular endosomes/bodies (MVB). MVB sorting primarily uses ubiquitin chains that are linked via lysine-63 of ubiquitin, which binds to a number of endosomal proteins that cluster and sort ubiquitinated membrane proteins into intralumenal vesicles. These intralumenal vesicles can either be delivered to the interior Lysosomes and be degraded, or they can be secreted as extracellular vesicles/exosomes, that carry and delivery contents to other cells to elicit a range biological effects. Many critical questions remain outstanding about how proteins are sorted into endosomal intralumenal vesicles and what differentiates how they are sorted to Lysosomes versus being sorted into exosomes. Aim1 will address these questions by examining the molecular mechanisms that two homologous sorting proteins use to differentially sort proteins into intralumenal vesicles that go to Lysosomes or become exosomes. Additional questions remain about whether ubiquitin attachment to membrane proteins in the trans- Golgi Network, Endosomes, and cell surface that have made it through their initial synthesis in the endoplasmic reticulum (so-called ‘post-ER’ proteins) exclusively sends them through the MVB pathway as their mode of degradation. We found that post-ER proteins modified with a different type of polyubiquitin chain are sent along a different pathway for degradation. Aim2 will study the molecular features of this pathway and assess how this pathway may contribute to controlling the quality and quantity of membrane proteins.