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 presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

Methods
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, Avanti Polar Lipids), 10 pmol t-Cer (18:0, Avanti Polar Lipids) and 50 pmol ergosteryl ester (15:0 and 19:0). The lipids in the organic phase were dried under a gentle stream of nitrogen at 37°C. Lipids were resuspended in 10 mM methanolic ammonium acetate and transferred to 96-well plates (Eppendorf Twintec 96). Mass spectrometry was performed on a Sciex QTRAP 6500+ mass spectrometer, equipped with chip-based (HD-D ESI Chip; Advion Biosciences) nano-electrospray infusion, and ionization (TriVersa NanoMate; Advion Biosciences; ​Özbalci et al., 2013​). The following precursor ion (PREC) or neutral loss (NL) scanning modes were used: +PREC184 (PC), +PREC282 (t-Cer), +NL141 (PE), +NL185 (PS), +NL277 (PI), +NL189 (PG), +NL115 (PA), +NL77 (ergosterol), +PREC379 (ergosteryl ester). Mass spectrometric analysis of ergosterol was performed by applying one-step chemical derivatization to ergosterol acetate in the presence of 300 pmol (first and second replicate) or 100 pmol (third and fourth replicate) of the internal standard (22E)-Stigmasta-5,7,22-trien-3-beta-ol (R202967; Sigma-Aldrich) using 100 μl acetic anhydride/chloroform (1:12 v/v) overnight under argon atmosphere according to (​Ejsing et al., 2009​). CL and MLCL quantification was done as described (Chowdhury et al., 2018). Data evaluation was done using LipidView (Sciex) and ShinyLipids (in-house developed software). DAG and TAG analysis was performed as described (Özbalci et al., 2013).

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