Scaffold Nups form two inner rings and two outer rings, which serve as the NPC structural core [4C7], and associate with the membrane through interactions with transmembrane Nups [3,8,9]. nuclear envelope. (B) IEM of co-expressed GFP-spNup131 Afatinib dimaleate and spMis6-GFP. A representative image is shown. Arrows indicate immunogold at the nuclear pores. The yellow-lined regions indicate immunogold near the SPB, corresponding to the signals from spMis6-GFP. (C, D) Immunoelectron micrographs of 20 nuclear pores used to generate the montage picture and distribution analysis in Fig 1C. Scale bars, 200 nm. (C) IEM of GFP-spNup131 and spMis6-GFP. (D) IEM of GFP-spNup132.(TIF) pgen.1008061.s002.tif (2.9M) GUID:?8C870ED9-3DC5-40A3-A462-01B01E31B1FA S3 Fig: Affinity capture/mass spectrometry of GFP-spNup131 and GFP-spNup132. (A, B) Proteins bound to GFP-spNup131 and GFP-spNup132. Images of Coomassie-stained SDS-PAGE gels are shown. Dots indicate the positions of molecular weight marker proteins shown on the left. Each gel was cut at the positions shown by the horizontal lines on the gel image. Proteins that correspond to major bands in each gel fragment Afatinib dimaleate were deduced by LC/MS/MS analysis and are indicated on the right. The list on the right shows proteins specifically bound to GFP-spNup131 and GFP-spNup132, Nups, and abundant proteins ( 20 spectra). Protein names are colored by their subcellular localizations according to gene ontology data (Pombase: https://www.pombase.org/): magenta, cytoplasmic proteins; blue, nuclear proteins; black, proteins of other or unidentified localizations. (C) Venn diagram showing proteins bound to GFP-spNup131 and GFP-spNup132 identified by LC/MS/MS analysis. Protein names are colored by their subcellular localizations: magenta, cytoplasmic proteins; blue, nuclear proteins; black, proteins of other or unidentified localizations.(TIF) pgen.1008061.s003.tif (762K) GUID:?29C5683F-8287-4430-8F76-D2AC86E418C6 S4 Fig: FM images of spFar11-GFP in wild type, = 0.41, students t-test); the duration of meiosis PIK3CD II was 28.3 3.9 min in wild type and 28.4 4.4 min in = 0.96, students t-test). n.s. stands for no significant difference. Numbers of observed cells are indicated at the bottom.(TIF) pgen.1008061.s006.tif (84K) GUID:?6175E832-C13B-4A36-9026-E5347B13F2FA S7 Fig: Characterization of the strains used in Fig 6. (A) Detection of GFP fused protein fragments by Western blot. strains used in this study. (DOCX) pgen.1008061.s010.docx (36K) GUID:?A19D7A15-4BD6-4D07-9D69-C40EAA5907E9 S3 Table: Dilution ratios of primary and secondary antibodies used for IEM. (DOCX) pgen.1008061.s011.docx (24K) GUID:?D102DA76-2F8F-47D5-9848-2BA56533A04E S1 Dataset: Individual IEM Afatinib dimaleate images of 20 NPCs used for superimposed images of Fig 1C (spNup131-GFP and spNup132-GFP). (PDF) pgen.1008061.s012.pdf (685K) GUID:?D1E4FE31-0A2B-4931-9C24-D89B49715312 S2 Dataset: Values of the distance between mCherry-spNup132 and GFP-spNup131 and those between mCherry-spNup131 and GFP-spNup132 measured for Fig 1E. (XLSX) pgen.1008061.s013.xlsx (14K) GUID:?5B6DCEDF-E64D-4AD1-8E23-4269763C8C91 S3 Dataset: Individual IEM images of 20 NPCs used for superimposed images of Fig 2B (spFar8-GFP). (PDF) pgen.1008061.s014.pdf (246K) GUID:?B3EE26C9-F5D1-4400-8D15-A70035298CCE S4 Dataset: Individual IEM images of 20 NPCs and the projection image analyzed for Fig 3A (spNup211-GFP). (PDF) pgen.1008061.s015.pdf (454K) GUID:?31D1667F-86E1-4664-987D-0C8947AA2F60 S5 Dataset: Values of the maximum fluorescence intensity of spNup211-GFP in wild type, immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast. Author summary The nuclear pore complexes (NPCs) form gateways to transport intracellular molecules between the nucleus and the cytoplasm across the nuclear envelope. The Nup107-160 subcomplex, that forms nuclear and cytoplasmic outer rings, is a key complex responsible for building the NPC by symmetrical localization on the nuclear and cytoplasmic sides of the nuclear pore. This structural characteristic was found in various organisms including humans and budding yeasts, and therefore believed to be common among all eukaryotes. However, in this paper, we revealed an asymmetrical localization of the homologous components of the human Nup107-160 subcomplex in fission yeast by immunoelectron and fluorescence microscopic analyses: in this organism, the Nup107-160 subcomplex is split into two pieces, and each of.