Biochemical Studies of Molecular Mechanisms Underlying Endoplasmic Reticulum Membrane Fusion

Abstract

The endoplasmic reticulum (ER), the largest continuous membrane-bound organelle in eukaryotic cells, plays critical roles in many cellular processes, including protein synthesis, protein modification and sorting, lipid synthesis, and regulation of calcium homeostasis. The cellular functions of the ER require a myriad of proteins and unique ER structures, which are maintained by homotypic ER membrane fusion. Although the exact mechanisms for the formation and maintenance of the ER structures are yet to be clarified, the dynamin-like GTPase atlastin, an evolutionarily conserved protein family, is believed to mediate homotypic ER fusion at three-way junctions of the ER tubular network. In this dissertation, I focus on elucidating how the fusogenic activity of atlastins is regulated in eukaryotic cells to maintain the ER network using various in vitro assays. In Part Ⅰ, I describe regulatory mechanism of the yeast lunapark Lnp1p in yeast atlastins Sey1p-mediated membrane fusion by using a newly developed assay for monitoring the formation of trans-Sey1p complexes between two membranes and in vitro fusion assay. This part dissects the molecular mechanisms of Lnp1p, a negative regulator of Sey1p-mediated ER fusion. In Part Ⅱ, using various fusion assays, I investigated the molecular regulatory mechanisms of human atlastins (ATL1, ATL2, ATL3) in membrane fusion. This part Ⅱ identified different fusion activities of human atlastins despite the similarities between ATL1, ATL2, and ATL3. M1-spastin, a neuron-specific protein, markedly increased ATL1-mediated liposome fusion, suggesting that it is a positive regulator of ATL1-mediated membrane fusion. On the other hand, the fusogenic activity of ATL2 is regulated by its C-terminal 39 amino acids (CH), indicating that the CH plays an essential role in the regulatory mechanisms. Intriguingly, however, ATL3 or cytosolic factors relieve the autoinhibition by the CH, enabling ATL2 to support ER fusion in vivo. Collectively, this study provides insight into the regulatory mechanisms of atlastin-mediated ER membrane fusion for the formation and maintenance of ER structure in cells.