Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex (original) (raw)

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Affinity capture of the trypanosome NPC and identification of new Nups.

(A) Schematic of the eukaryotic phylogenetic tree, adapted from Field et al., 2014 [38], highlighting the close evolutionary distance between yeast and humans versus more divergent eukaryotes such as trypanosomes (Excavates). SAR and CCTH correspond to Stramenoplies, Apicomplexa, Rhizaria and Cryptophyta, Centrophelida, Telonimia, Haptophyta, respectively. FECA and LECA refer to the first and last eukaryotic common ancestors. (B) Using the green fluorescent protein (GFP)-tagged nuclear basket protein Nup110 (marked with a ‡), we affinity isolated structural components of the NPC (dark grey), FG repeat containing Nups (green), and specifically associated proteins (light grey), which include transport factors and the major trypanosome lamina protein NUP-1. Affinity isolates were resolved by SDS-PAGE and visualized by Coomassie staining. Protein bands were excised and identified by mass spectrometry (MS). We discovered five new nucleoporins (in bold); assignments are based on secondary structure prediction and localization, as well as multiple pullouts that indicate bona fide association with trypanosome NPC components. Putative nuclear envelope proteins, α/β tubulin, and known contaminants (immunoglobulin heavy chain, variant VHH, and light chains of polyclonal llama anti-GFP antibodies) are marked by asterisks. A comprehensive list of all proteins identified is shown in S1 Fig. A schematic of the NPC is shown to highlight the architecture of the NPC, based on the Saccharomyces cerevisiae quaternary structure. Grey and green shapes represent core scaffold Nups and FG-Nups, respectively, identified by DeGrasse et al., 2009 [27]. White shapes represent subcomplexes for which components were not identified in that earlier proteomic screen. (C) Direct visualization of the GFP-tagged newly identified Nups confirms that they exhibit the punctate nuclear rim localization characteristic of NPCs. The corresponding 4’, 6-diamino-2-phenylindoledihidrochloride (DAPI) fluorescence was used to image the DNA (k = kinetoplast, n = nucleus). (D) Secondary structure features and fold prediction of the five newly identified Nups. The _y_-axis indicates the confidence score of the predicted secondary structure element. Models of fold types are shown on the right, together with potential opisthokont orthologs based on the predicted fold types. RRM, RNA recognition motif; TM, _trans_-membrane domain. Fold models are based on PDB structures: 1XIP (β-propeller of Nup159), 3P3D (RRM of Nup35), 2KA2 (TM), 1AQ5 (coiled coil), and 4MHC (α-solenoid of Nup192). TbNup152 is approximately 153 kDa but has been assigned 152 to prevent confusion with the well-studied human Nup153.

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doi: https://doi.org/10.1371/journal.pbio.1002365.g001