Insertion of the nuclear pore complex and the yeast spindle pole body into the nuclear envelope.

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Here, we will investigate how large proteinaceous structures become inserted into the double membrane of the nuclear envelope (NE). This is still one of the big open questions in cell biology. We will address this using the nuclear pore complex (NPC) and the spindle pole body (SPB), the yeast centrosome, from budding and fission yeast as models. The emerging picture is that NPCs assemble in interphase from within the nucleus. Nucleoporins accumulate underneath the inner nuclear membrane (INM), deform this membrane and eventually the INM and outer nuclear membrane (ONM) fuse. Using budding yeast, we recently identified the paralogues Brr6/Brl1, two integral membrane proteins of the NE, as components of NPC biogenesis sites that function late in NPC biogenesis, perhaps in INM/ONM fusion. Starting with these two proteins, we aim to understand how NPCs assemble in the intact NE. We will test the functions of budding yeast Brr6/Brl1 by genetic and biochemical approaches. In particular, we will insert purified Brr6/Brl1 into proteo-liposomes and measure their properties. Analysis of local lipid changes using lipid sensors will indicate whether Brr6/Brl1 modulate lipids during NPC biogenesis. Interestingly, our data suggest that the NE insertion of the budding yeast SPB requires the assistance of NPCs that dock onto the growing daughter SPB (dSPB) that first sits on the cytoplasmic face at the ONM. These NPCs may provide the ONM/INM fusion site into which the dSPB is inserted or deliver shared factors of NPCs/SPBs (Ndc1) to the dSPB. We will analyze SPB duplication by electron tomography in order to see the relationship between dSPB and NPCs. Super resolution live cell imaging will be used to clarify the fate of the NPCs close to the dSPB. Next, budding yeast NPC/SPB biogenesis will be compared with fission yeast. Surprisingly, the fission yeast homologue SpBrr6 (fission yeast lacks BRL1) is needed for the insertion of the SPBs into the NE while our data argue against such a function of Brr6/Brl1 in budding yeast. Whether SpBrr6 plays a role in NPC biogenesis is unclear. To understand the general functions of Brr6-like proteins, it is important to know whether SpBrr6 also has a role in NPC biogenesis. Immunoelectron microscopy will indicate the localization of fission yeast SpBrr6 with NPCs and SPBs. Electron microscopy of conditional lethal Spbrr6(ts) cells will help to understand the functions of SpBrr6 during NE insertion of the SPB and NPC biogenesis. Taken together, this proposal will clarify how the NE is modulated in order to allow SPB insertion and NPC biogenesis.