Molecular Mechanisms of the Formation and Maintenance of the Tubular Endoplasmic Reticulum Network
Powers, Robert Edward
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AbstractMembrane-bound organelles, a defining feature of eukaryotic cells, display a diverse set of characteristic shapes that range from highly spherical, to flattened sheets, and even thin tubules. Therefore, how the characteristic shape of an organelle is generated, maintained, and modified is a fundamental question in eukaryotic cell biology. A powerful model system for studying organelle morphology is the endoplasmic reticulum (ER), which consists of a network of membrane sheets and tubules that extend throughout a cell. Previous studies have shown that the high membrane curvature of the tubules is generated and stabilized by integral membrane proteins of the reticulon and Yop1/REEP families and that individual tubules are fused together by the dynamin-like GTPases Atlastin (in metazoans) and Sey1p/RHD3 (yeast/plants). Although an in vitro assay for ER network formation has been developed using Xenopus egg extracts, the minimal set of components needed to form a tubular ER network has not been identified, and whether these minimal components allow for the ER dynamics observed in vivo is not known.
In this thesis, I will focus on the molecular mechanisms responsible for shaping the endoplasmic reticulum and offer insight into how these mechanisms give rise to the distinct tubular architecture observed for particular subdomains of the endoplasmic reticulum. I demonstrate that the minimal set of proteins needed to form the tubular ER network consists solely of a curvature-stabilizing protein and a membrane-fusing protein. Co-reconstitution of Saccharomyces cerevisiae Sey1p with a number of different curvature-stabilizing proteins of the reticulon and Yop1/REEP families yield proteoliposomes that, when incubated with GTP, form tubular networks that are nearly indistinguishable from those observed in the extracts of Xenopus laevis eggs. Furthermore, these reconstituted networks have the same dynamic behaviors as ER networks in cells, including junction sliding and ring closure. Finally, the integrity of the synthetic network is dependent upon the GTPase activity of the membrane-fusing protein, as incubation of pre-formed reconstituted networks with GTPγS leads to rapid network disassembly. Taken together, these results demonstrate that the tubular ER can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.
I also describe my initial steps toward obtaining a structure of a curvature-stabilizing protein of the Yop1/REEP family using x-ray crystallography. The lack of an atomic-resolution structure of any of these proteins has left their exact mechanism of curvature generation and stabilization unknown. Unfortunately, given their small size and lack of hydrophilic surfaces, the Yop1/REEP proteins represent a difficult target for structural studies. To this end, I sought to use new tools that have been adapted for membrane protein crystallization to attempt to obtain a structure of the protein REEP5. These tools included lipidic cubic phase crystallization (LCP) techniques, as well as nanobody-aided crystallization. While initial LCP crystallization experiments failed to yield any crystals, I successfully isolated a number of nanobodies that bind to REEP5 with high affinity using an in vitro yeast display system. These nanobodies will be useful in future crystallization as well as in vitro biochemical experiments aimed at understanding REEP5 function, and more broadly, at how the reticulon and Yop1/REEP proteins generate and stabilize membrane curvature.
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