Conservation and divergence at the nuclear envelope.

<p>The major protein and nucleic acid complexes responsible for control of gene expression, nucleocytoplasmic transport, and regulation of nuclear architecture are shown. The circular nucleus diagram is divided into three colourised sectors that correspond to those of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006170#ppat.1006170.g001" target="_blank">Fig 1</a>. Elements are colourised that are known to deviate from likely LECA components, whilst unknown elements are shown as open symbols. Mixed purple/green is used to designate factors that are shared between Opisthokonts and Apicomplexa. Significantly, the extensively studied <i>Homo sapiens</i> nucleus appears to retain much of the machinery of the LECA, whilst trypanosomes have several clear examples of divergent molecular systems that subtend nuclear functions. In Apicomplexa, the basic nuclear system appears once more to be similar to the LECA, although several aspects (for example, the composition of the nuclear pore complex and the identity of the lamina) remain unknown at this time; evidence suggests that Apicomplexa do not possess a LECA/mammalian type lamina, suggesting the presence of a novel machinery awaiting discovery.</p

Overview of eukaryotic phylogeny emphasising the supergroup affiliation of organisms discussed here.

<p>Each of five recognised eukaryotic supergroups is shown as a coloured triangle to indicate that it contains a great many lineages, which are under continual diversification; groups not discussed are in gray, whilst Excavata (teal), stramenopiles, alveolates, and Rhizaria (SAR, red), and Opisthokonta (purple) are shown with icons for representative organisms. All of these groups radiated rapidly following the origin of eukaryotes and evolution of the LECA. Relationships are based on recent views of the branching order but should not be considered definitive.</p

A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies

The central players in most cellular events are assemblies of macromolecules. Structural and functional characterization of these assemblies requires knowledge of their subunit stoichiometry and intersubunit connectivity. One of the most direct means for acquiring such information is so-called “native mass spectrometry (MS)”, wherein the masses of the intact assemblies and parts thereof are accurately determined. It is of particular interest to apply native MS to the study of endogenous protein assembliesi.e., those wherein the component proteins are expressed at endogenous levels in their natural functional states, rather than the overexpressed (sometimes partial) constructs commonly employed in classical structural studies, whose assembly can introduce stoichiometry artifacts and other unwanted effects. To date, the application of native MS to the elucidation of endogenous protein complexes has been limited by the difficulty in obtaining pristine cell-derived assemblies at sufficiently high concentrations for effective analysis. Here, to address this challenge, we present a robust workflow that couples rapid and efficient affinity isolation of endogenous protein complexes with a sensitive native MS readout. The resulting workflow has the potential to provide a wealth of data on the stoichiometry and intersubunit connectivity of endogenous protein assembliesinformation that is key to successful integrative structural elucidation of biological systems

Affinity isolation of TbNPC subcomplexes.

<p>TbNup nomenclature has been shortened to NupX, with subsequent comigrating Nups simply given their identification number that corresponds to their molecular weight, with the exception of Sec13 (i.e, Nup158, 152 instead of TbNup158, TbNup152). (A) Coomassie-stained SDS-PAGE of GFP-tagged members of the inner ring of the TbNPC. Predicted homologs, predicted fold types, and the GFP-tagged Nup are shown above each gel. The affinity handle (blue ‡) and isolated proteins identified by mass spectrometry are shown on the right of each protein gel. The asterisks designate known contaminants and non-NPC/nuclear envelope proteins as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.g001" target="_blank">Fig 1</a>. Full lists are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.s001" target="_blank">S1 Fig</a>. Nup225, 181, 96, 62, 53a, and 53b form a distinct complex with each other. Nup62 exists as two proteins of different sizes that probably reflect allelic variation due to expansion or contraction of FG-repeats. Nup65 associates with Nup96 and 225. Nup144 weakly interacts with Nup89, whilst Nup119 associates with multiple nuclear pore subcomplexes. (B) Affinity isolated members of the outer ring of the TbNPC. Most of the Nups associate with each other, with a few minor exceptions. Nup109 associates weakly with the rest of the complex and is lost in most affinity capture conditions. However, it is a bona fide member of the outer ring, as it affinity isolates the corresponding members of the Nup89 complex. The Nup89 complex also interacts with the lamin analog NUP-1 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref042" target="_blank">42</a>], the nuclear basket Nup110 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref027" target="_blank">27</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref035" target="_blank">35</a>], and the FG-Nup98. The presence of Sec13 in both the NPC and COPII complex is highlighted by the affinity capture of Nups as well as the abundant Sec31, a vesicle coat protein that forms a heterotetramer with Sec13 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref043" target="_blank">43</a>], when Sec13-GFP is used as the affinity handle. (C) FG-Nup64 and 98 associate with multiple NPC subcomplexes. Nup75 only interacts with Nup64 and 98, suggesting a close association of these three FG-Nups. (D) Affinity isolation of Nup76 and several FG-Nups with their interacting partners. Nup76 associates with FG-Nups 140, 149, and several members of the outer ring complex. Additionally, the mRNA export factor Mex67 associates with this subcomplex.</p

Membrane anchoring and the core module of the TbNup89 complex.

<p>(A) TbNup65 is a TM containing protein. (i) Western blot showing sodium carbonate extraction of TM proteins [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref064" target="_blank">64</a>], confirming that TbNup65 and Tb927.4.4760—a nuclear envelope and Golgi marker protein—are TM proteins, as they are predominantly recovered in the pellet (Pel) whilst the non-TM α-solenoid TbNup89 is predominantly recovered in the supernatant (Sup). (ii) An illustration of the predicted secondary structure and the differences in nuclear membrane interaction between TbNup65 and its yeast, human, and plant orthologs (ScNup53, HsNup35, and AtNup35, respectively). The opisthokont and plant Nup53/35 are mainly disordered (Disopred), unlike the trypanosome Nup65 that has several structured regions. (B) The Nup89 complex is comprised of eight proteins (including TbNup109) that can be further reduced into a core module consisting of just four proteins when the stringency of the extraction buffer is increased. A schematic of the outer ring as well as subcomplexes is shown. Nup41 and Sec13 are beta propellers, Nup82 and 89 are alpha solenoids, Nup109, 132 and 152 are beta/alphas and Nup158 is a FG-Nup/alpha solenoid.</p

Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.

More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors--e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs--remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm.</p

Model of the TbNPC and a putative role of Ran in mRNA export.

<p>(A) A model of the TbNPC compared to the yeast NPC. Only one copy of the inner ring is illustrated for simplicity. The anchoring mechanism of the TbNPC is provided by a single inner ring Nup (TbNup65) that in yeast (ScNup53/59) interacts with the NE via an ALPS motif. Trypanosomes lack the whole pore membrane ring comprised of Pom152 (GP210 in humans and plants), Pom34, and NDC1 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref005" target="_blank">5</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref006" target="_blank">6</a>]. The TbNPC is largely symmetric, with asymmetry provided by its nucleoplasmic interactions through two nuclear basket Nups that are half the size of their opisthokont analogs [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref035" target="_blank">35</a>]. Significantly, there are no clear orthologs of Dbp5 and Gle1, coincident with the lack of cytoplasmic or nucleoplasmic biased FG-Nups in trypanosomes. Instead, TbNup76, the candidate ortholog of the cytoplasm-specific Nup82/88 in opisthokonts, localizes to both faces of the NPC. (B) Left, model highlighting the conserved inner ring core (blue) and differences in asymmetry (red) in excavates and opisthokonts as represented by trypanosomes and yeast. Orthologs of cytoplasmic Nups or mRNA remodeling factors are absent from trypanosomes. Right, affinity capture of the conserved nonkaryopherin RNA exporter Mex67 co-isolates Ran, suggesting a putative role for the GTPase Ran in mRNA export in trypanosomes (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.g006" target="_blank">Fig 6A</a>). Bulk polyA mRNA export in opisthokonts is driven by ATP through the actions of the ATP-dependent DEAD box helicase DBP5, RNA export factor Gle1, and inositol hexakisphosphate (IP₆) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref022" target="_blank">22</a>].</p

Determination of the relative NPC location of each subcomplex.

<p>(A) Immunogold electron localization of GFP-tagged Nups using polyclonal anti-GFP rabbit antibodies to determine relative positions of Nups within the TbNPC (Methods). We picked NPCs sectioned perpendicular to the NE plane, selected a radius of 300 nm around the estimated center of each NPC, and excised each image (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.s002" target="_blank">S2 Fig</a>). We then aligned and created a superimposed montage of several excised NPC images [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref006" target="_blank">6</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref058" target="_blank">58</a>]. Graduated lines adjacent to each iEM montage are scaled to represent distances of 50 nm. Major features of each montage are represented in the illustration on the right: NE, nuclear envelope; NPC, nuclear pore complex; N, nucleoplasm; and C, cytoplasm. (B) Statistical analysis of relative locations of select TbNups within the TbNPC, based on the distribution of gold particles from various iEM montages. X and Y positions of gold particles from iEM montages for each selected Nup were measured, from which the Z- and R- (cylindrical rotational axis of the NPC) axes were calculated and displayed in a tabulated form (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.s003" target="_blank">S3 Fig</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.s009" target="_blank">S1 File</a>, and the full table in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.s011" target="_blank">S2 Table</a>). Z average values are positive or negative to represent localizations above and below the midplane of the NPC. TbNup110 only has a negative value, as it clearly localizes to the nucleoplasm only. Abbreviations: ave (average), Err (error), N(R) (number of gold particles used to calculate the R-axis), N(Z) (number of gold particles used to calculate the Z-axis), NPCs (number of NPCs used to generate either the N(R) or N(Z) for each selected TbNup). (C) Illustrated representation of the relative position of each Nup within the TbNPC. Nup64, 98, and Nup119 are centrally located, whereas Nup62, 76, and 89 appear to be positioned further away from the central channel. The nuclear basket TbNup110 has a clear nucleoplasmic localization. R- and Z-axes errors are plotted based on the 95% level of a peak finding algorithm [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref006" target="_blank">6</a>].</p

A comparison of the individual units of the evolutionarily conserved outer ring complex between trypanosomes, opisthokonts, and plants.

<p>A comparison of the individual units of the evolutionarily conserved outer ring complex between trypanosomes, opisthokonts, and plants.</p

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

<p>(A) Schematic of the eukaryotic phylogenetic tree, adapted from Field et al., 2014 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref038" target="_blank">38</a>], 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 V_HH, and light chains of polyclonal llama anti-GFP antibodies) are marked by asterisks. A comprehensive list of all proteins identified is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.s001" target="_blank">S1 Fig</a>. A schematic of the NPC is shown to highlight the architecture of the NPC, based on the <i>Saccharomyces cerevisiae</i> quaternary structure. Grey and green shapes represent core scaffold Nups and FG-Nups, respectively, identified by DeGrasse et al., 2009 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002365#pbio.1002365.ref027" target="_blank">27</a>]. 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 <i>y</i>-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, <i>trans</i>-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.</p