ER and post-ER quality control of integral membrane proteins - Approximately one-quarter of all proteins synthesized in human cells integrate into the membrane of the endoplasmic reticulum (ER) as they are translated. However, membrane protein folding is problematic. First, structurally diverse integral membrane proteins face the challenge of folding co-translationally and post- translationally in three chemically distinct environments: the ER lumen, the ER membrane, and the cytoplasm. Second, many of these transmembrane domain (TMD)-containing proteins transport ions and other hydrophilic solutes, so sequences within TMDs favor amino acids containing charged and polar side chains. While these amino acids facilitate substrate transport, they are also inherently unstable in the membrane. And third, most ion channels and transporters are oligomeric, so each subunit must find its partner(s) after membrane insertion. As a result of these hurdles, membrane proteins fold inefficiently, and inherited mutations can further compromise folding efficiency. To overcome toxic effects arising from misfolded protein accumulation, the ER is equipped with a pathway to remove nonnative and incompletely assembled species. This disposal pathway, first reconstituted and named by the PI, is known as ER associated degradation (ERAD). To date, nearly 70 substrates, most of which are membrane proteins, are linked to various diseases and arise from the targeted destruction of misfolded proteins in the ER. Since its discovery, the PI’s long term research objectives have been to dissect individual steps during ERAD. This pursuit has focused on the molecular mechanisms underlying substrate selection, retrotranslocation from the ER into the cytosol, ubiquitination, and degradation by the 26S proteasome. In parallel, the Brodsky lab has helped define the molecular etiology of nearly a dozen diseases. Yet, critical unanswered questions in the ERAD field remain, including: (1) How does the ERAD pathway handle aggregation-prone membrane proteins, which might be difficult to retrotranslocate from the ER membrane? (2) Are other pathways required to eliminate these toxic aggregated species? (3) What is the mechanism that prevents misfolded integral membrane ERAD substrates from trafficking beyond the ER? (4) Can “new” mutant alleles in human genes that encode misfolded ion channels be rapidly identified and characterized? This last question reflects the first step toward developing personalized therapies for loss-of- function ERAD-associated diseases. To answer these questions, the lab will capitalize on emerging techniques in diverse model systems, each with unique strengths, as well as ongoing collaborations. Innovative tools, which are new to the field, will also be used. Together, the proposed research objectives lie at the heart of the lab’s long-term research goals, will address critical knowledge gaps, employ experimentally rigorous and transdisciplinary strategies, provide the research community with novel methods to address other unanswered questions in cell biology, and lead to the generation of new testable hypotheses.
