Lipid analysis of S.c. cells expressing ProtA-ScNdc1 (261-655), ProtA-AHNdc1-ProtA, or ProtA-ScHmg1

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, and few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1’s transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1’s C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1’s amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and p..., Yeast cells were grown overnight in corresponding media, harvested in mid-exponential growth phase, and resuspended in 50 mM HEPES pH 7.4 containing 0.8 mg/ml Zymolyase 100T (Carl Roth). Cells were mechanically lysed using glass beads. Lipidomics analyses were performed by a shotgun approach as recently described (Papagiannidis et al., 2021). Lipid extracts were prepared in the presence of internal lipid standards via acidic Bligh-Dyer lipid extraction (Bligh and Dyer, 1959). The used master mix of lipid internal standards contained 50 pmol d7-PC mix (15:0/18:1-d7, Avanti Polar Lipids), 25 pmol PI (17:0/20:4, Avanti Polar Lipids), 50 pmol PE and 10 pmol PS (14:1/14:1, 20:1/20:1, 22:1/22:1, semi-synthesized as described in (Ozbalci et al., 2013)), 15 pmol PA (PA 17:0/20:4, Avanti Polar Lipids), 10 pmol PG (14:1/14:1, 20:1/20:1, 22:1/22:1), semi-synthesized as described in (Ozbalci et al., 2013)), 40 pmol DAG (17:0/17:0, Larodan), 40 pmol TAG (D7-TAG-Mix, LM-6000/D5-TAG 17:0,17:1,17:1, Av...,