Translate Membrane Structure Into Protein Functions.

Pennsylvania State Univ Hershey Med Ctr, Hershey PA

Investigators

Abstract

Abstract Membrane geometry is generated and maintained by the interplay of protein-lipid and lipid-lipid interactions. Detection and remodeling of membrane shapes are part of many essential cellular processes. Furthermore, an increasing number of proteins have been found to depend on specific membrane geometry for their function (e.g. ArfGAP1 and Atg3) and localization (e.g. SpoVM). We are investigating the structural and molecular mechanisms for the recognition of membrane geometry and for the translation of membrane geometry into protein function. While pursuing these goals, we are developing innovative tools for the study of geometry-sensitive molecules in native-like environments using spherical nanoparticle supported lipid bilayers and membrane vesicles directly isolated from cells with an in situ NMR spectroscopy. Autophagy is a conserved stress-response pathway in eukaryotes. De novo formation of autophagosomes to engulf cargos targeted for degradation is the hallmark of autophagy. This process is membrane-remodeling intensive and requires over 30 proteins, but how their actions are spatially and temporally coordinated is a major knowledge gap. Atg3 catalyzes a direct covalent conjugation of LC3 to the amino group of phosphatidyl- ethanolamine (PE) lipid. The conjugate LC3–PE triggers phagophore expansion and acts as an adaptor for sequestering cargos for breakdown. Our study has revealed that selective binding of human Atg3 (hAtg3) to highly curved membranes is tightly linked to its conjugase activity via a multifaceted membrane association mechanism. In this system, the highly curved membrane functions like a classical E3 ligase by bringing substrates (Atg3–LC3 and PE lipids) into proximity and priming the active site of Atg3 for catalysis. These results have made major contributions to establishing and advancing the concept that the distinct membrane structure of the cup-like phagophore spatiotemporally regulates autophagosome biogenesis. We are determining the structural and molecular basis of how the multifaceted membrane association of hAtg3 coordinates its curvature sensitivity and conjugase activity to promote LC3–PE conjugation only on the target membrane in the context of the putative E3-like Atg12-Atg5/Atg16 complex. My collaborator, Dr. Wang, has recently discovered that phagophore closure requires a subset of ESCRT units including VPS37A, CHMP2A, and Vps4. ESCRTs mediate membrane remodeling and scission throughout the cell. We have demonstrated that recognition of highly curved membranes by two hydrophobic motifs in the VPS37A is essential for its phagophore localization and autophagosome closure. We are investigating how the VPS37A UEVL domain, in conjunction with the two membrane binding motifs, orchestrates the spatiotemporal process of autophagosome closure. Results from our study will provide the necessary foundation for continued analyzing the poorly understood biogenesis of the autophagosome during autophagy and, moreover, shed lights on the interplay between membrane structure and protein function.