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Abstract

The nuclear envelope (NE) is a hallmark of eukaryotic cells, functioning as a physical barrier that separates the genome from the cytosol. In organisms with closed mitosis, essential macromolecular complexes like the nuclear pore complex (NPC) and the spindle pole body (SPB) must embed into the NE, requiring the fusion of both the inner (INM) and outer nuclear membranes (ONM). How this fusion of the two membrane bilayers happens remains unclear. In this study, I report that three integral membrane proteins - Apq12, Brl1 and Brr6 - are required in NPC biogenesis by associating with the assembly sites in the NE during the interphase assembly process. I show that Apq12, Brl1 and Brr6 localize to the NE and endoplasmic reticulum (ER). Each protein carries two transmembrane domains that flank an amphipathic helix (AαH) that is located in the perinuclear space. These amphipathic helices have the ability to localize to the nuclear envelope on their own. In the first part, I report that the integral membrane protein Apq12 plays a crucial role in modulating the NE/ER and cooperates with NPC biogenesis factors Brl1 and Brr6. Disruption of its AαH (apq12-ah) leads to defects in NPC biogenesis and NE integrity, while not affecting the topology or localization of Apq12-ah within the NE/ER. Moreover, overexpression of wild-type APQ12, but not the apq12-ah, induces pronounced proliferation of the ONM/ER and leads to enrichment of phosphatidic acid (PA) at the NE. Both Apq12 and Apq12-ah are found to associate with NPC biogenesis intermediates, with the mutation of the AαH resulting in elevated levels of Brl1 and increased interaction between Brl1 and Brr6. In the second part, I show that not only is the AαH of Brl1 essential for its function, but over-expression of its mutant results in the formation of a novel type of herniation; petal- like structures, resulting from failed INM-ONM fusion. Furthermore, it also leads to mislocalization of a number of cytoplasmic nucleoporins, consistent with the inside-out model of interphase NPC assembly. Lastly, I show that Brr6 also has a role in INM-ONM fusion as ts-mutants with mutations in the cysteine residues lead to the formation of herniations. Over-expression of its AαH mutant brr6L145E causes mislocalization of all nucleoporins except the transmembrane nucleoporins, suggesting an additional role beyond INM-ONM fusion during biogenesis. Taken together, this work sheds light on the process of membrane fusion during NPC biogenesis. The data shows that the amphipathic α-helix of Apq12 plays a critical role in regulating the functions of Brl1 and Brr6, potentially facilitating PA accumulation at the NE making it more susceptible to deformations. Brl1 and Brr6 also localize to the sites of NPC assembly on the INM and ONM, respectively, and bring the two bilayers together for fusion by interactions between their anti-parallel helix bundles stabilized by two disulfide bridges (DAH). This study offers a conceptual framework for understanding the fusion of the nuclear envelope during NPC biogenesis. It is highly probable that the fundamental principles are conserved across yeast and humans. Further comparative analysis of the assembly process in other organisms will elucidate these conserved principles.