DOI: 10.5061/dryad.s7h44j1bw
title: Lipid analysis of S.c. cells expressing ProtA-ScNdc1 (261-655), ProtA-AHNdc1-ProtA, or ProtA-ScHmg1

description: 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.
description: 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). 
 Bligh, E.G., and W.J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911–917. 10.1139/y59-099 Ejsing, C.S., J.L. Sampaio, V. Surendranath, E. Duchoslav, K. Ekroos, R.W. Klemm, K. Simons, and A. Shevchenko. 2009. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. Proc. Natl. Acad. Sci. USA. 106:2136–2141. 10.1073/pnas.0811700106 Özbalci, C., T. Sachsenheimer, and B. Brügger. 2013. Quantitative analysis of cellular lipids by nano-electrospray ionization mass spectrometry. Methods Mol. Biol. 1033:3–20. 10.1007/978-1-62703-487-6_1 Papagiannidis, D., P.W. Bircham, C. Lüchtenborg, O. Pajonk, G. Ruffini, B. Brügger, and S. Schuck. 2021. Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex. EMBO J. 40. e107958. 10.15252/embj.2021107958 Chowdhury A, Aich A, Jain G, Wozny K, Lüchtenborg C, Hartmann M, Bernhard O, Balleiniger M, Alfar EA, Zieseniss A, Toischer K, Guan K, Rizzoli SO, Brügger B, Fischer A, Katschinski DM, Rehling P, Dudek J. Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1α Expression in Hypoxia. Cell Rep. 2018 Oct 16;25(3):561-570.e6. doi: 10.1016/j.celrep.2018.09.057.