Dissecting the molecular mechanisms of membrane protein complex assembly

About This Grant

PROJECT SUMMARY This project aims to uncover the molecular mechanisms that regulate membrane protein complex assembly at the human endoplasmic reticulum (ER) membrane. While significant progress has been made in understanding membrane protein insertion and folding, little is known about how thousands of membrane proteins find their correct binding partners to assemble into functional complexes of defined stoichiometry. Based on our preliminary data and published work, we hypothesize that assembly is highly regulated, and we plan to uncover the physiological roles of the underlying regulatory mechanisms using the large family of oligomeric voltage- gated ion channels (VGICs) as model complexes. VGICs fulfill many essential functions and for example mediate excitation-contraction coupling in heart and muscle cells, and trigger hormone and neurotransmitter release in secretory and neuronal cells. Mutations that impair VGIC biogenesis or function cause severe cardiological, neuropsychiatric, and neurodevelopmental diseases, emphasizing a critical need to better understand the assembly and quality control pathways that control their cellular levels. Our approach combines genetics, cell biology, and structural biology to determine the physiological role and mechanism of action of two poorly characterized VGIC assembly factors and to identify and characterize novel VGIC assembly factors. We will begin by advancing our understanding of voltage-gated calcium channel assembly by dissecting a potential novel chaperone function of the ER membrane protein complex (EMC). Using fluorescent calcium channel reporter cell lines and function-separating mutations, we have shown that EMC’s novel assembly function is required for calcium channel assembly in human cells. By reconstituting early calcium channel assembly events using an in vitro translation and ER insertion system, we will analyze how the EMC protects nascent channels from promiscuous interactions, aggregation and premature degradation to facilitate assembly. Additionally, we have developed selective nanobody inhibitors to investigate the physiological relevance of EMC’s novel assembly factor function in human iPSC-derived cardiomyocytes. These selective tools and assays now enable us to explore EMC’s assembly client spectrum using mass spectrometry. In parallel, we will investigate the assembly and quality control of other VGICs, such as potassium channels, using genome-wide genetic screens and characterize candidate factors using our established pipeline. This research will yield critical insights into the regulatory processes governing membrane protein assembly and identify potential therapeutic targets for VGIC- related diseases.