Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans

To define the extent of the modification of the nuclear pore complex (NPC) during Aspergillus nidulans closed mitosis, a systematic analysis of nuclear transport genes has been completed. Thirty genes have been deleted defining 12 nonessential and 18 essential genes. Several of the nonessential deletions caused conditional phenotypes and self-sterility, whereas deletion of some essential genes caused defects in nuclear structure. Live cell imaging of endogenously tagged NPC proteins (Nups) revealed that during mitosis 14 predicted peripheral Nups, including all FG repeat Nups, disperse throughout the cell. A core mitotic NPC structure consisting of membrane Nups, all components of the An-Nup84 subcomplex, An-Nup170, and surprisingly, An-Gle1 remained throughout mitosis. We propose this minimal mitotic NPC core provides a conduit across the nuclear envelope and acts as a scaffold to which dispersed Nups return during mitotic exit. Further, unlike other dispersed Nups, An-Nup2 locates exclusively to mitotic chromatin, suggesting it may have a novel mitotic role in addition to its nuclear transport functions. Importantly, its deletion causes lethality and defects in DNA segregation. This work defines the dramatic changes in NPC composition during A. nidulans mitosis and provides insight into how NPC disassembly may be integrated with mitosis.     

Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos

Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.     

The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.     

核孔复合体的结构及其功能

核孔复合体(Nuclear pore complexes, NPCs)镶嵌在核膜上,是细胞核与细胞质之间的唯一通道。冷冻电子X射线断层扫描将环状NPCs分为三个环,分别称为胞质环、内环和核质环,胞质环上附有胞质纤丝,核质环上附有核篮。由于物种不同,NPCs由30～50多种不同的核孔蛋白(nucleoporins, Nups)组成,但结构和功能高度保守。根据其结构、氨基酸序列,NPCs定位和功能,Nups被分为跨膜Nups、屏障Nups、骨架Nups、胞质纤丝Nups和核篮Nups。相互间作用稳定、紧密连接的数个Nups可组成亚复合体。为了应对不同生理需要,NPCs处于高度动态变化中,间期和有丝分裂期均可通过组装和去组装改变核孔数量和功能。NPCs的主要功能是调控核质转运,小分子物质可自由扩散,大分子物质则需在核转位信号和转运载体的介导下以主动运输的方式进行转运。除了核质转运这一主要功能外,Nups还能以一个独立于转运的方式影响基因组功能。通过影响染色质结构和影响转录调控元件对靶基因的访问,Nups促进或抑制转录。在酵母,Nups介导的基因调控主要由位于NPCs中的Nups执行;在多细胞生物,不仅NPCs中的Nups,核质内游离的Nups也具有基因调控功能。此外,Nups还能通过参与形成染色质边界和形成转录记忆对基因进行调控。在增殖细胞, Nups通过与DNA修复机器相互作用,参与DNA损伤修复,保护基因组完整性。有丝分裂时,Nups协助核膜解体和中心体迁移,并通过作用于着丝粒来控制有丝分裂组件的空间定位与活性,稳定它们与微管之间的相互作用,保证纺锤体正常组装和染色体准确分离。总之,NPCs与生物分子的核质转运、基因表达和细胞周期密切相关,它的结构和功能的稳定是真核细胞生长、增殖、分化等生命活动的基本保证。     

Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .     

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.     

The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex

All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ∼60 MD in vertebrates, they are comprised of only ∼30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes.     

The SUN protein Mps3 controls Ndc1 distribution and function on the nuclear membrane

In closed mitotic systems such as Saccharomyces cerevisiae, nuclear pore complexes (NPCs) and the spindle pole body (SPB) must assemble into an intact nuclear envelope (NE). Ndc1 is a highly conserved integral membrane protein involved in insertion of both complexes. In this study, we show that Ndc1 interacts with the SUN domain–containing protein Mps3 on the NE in live yeast cells using fluorescence cross-correlation spectroscopy. Genetic and molecular analysis of a series of new ndc1 alleles allowed us to understand the role of Ndc1–Mps3 binding at the NE. We show that the ndc1-L562S allele is unable to associate specifically with Mps3 and find that this mutant is lethal due to a defect in SPB duplication. Unlike other ndc1 alleles, the growth and Mps3 binding defect of ndc1-L562S is fully suppressed by deletion of POM152, which encodes a NPC component. Based on our data we propose that the Ndc1–Mps3 interaction is important for controlling the distribution of Ndc1 between the NPC and SPB.     

The fission yeast Nup107-120 complex functionally interacts with the small GTPase Ran/Spi1 and is required for mRNA export, nuclear pore distribution, and proper cell division

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.     

A Highlights from MBoC Selection: Tts1, the fission yeast homologue of the TMEM33 family,functions in NE remodeling during mitosis

The fission yeast Schizosaccharomyces pombe undergoes “closed” mitosis in which the nuclear envelope (NE) stays intact throughout chromosome segregation. Here we show that Tts1, the fission yeast TMEM33 protein that was previously implicated in organizing the peripheral endoplasmic reticulum (ER), also functions in remodeling the NE during mitosis. Tts1 promotes insertion of spindle pole bodies (SPBs) in the NE at the onset of mitosis and modulates distribution of the nuclear pore complexes (NPCs) during mitotic NE expansion. Structural features that drive partitioning of Tts1 to the high-curvature ER domains are crucial for both aspects of its function. An amphipathic helix located at the C-terminus of Tts1 is important for ER shaping and modulating the mitotic NPC distribution. Of interest, the evolutionarily conserved residues at the luminal interface of the third transmembrane region function specifically in promoting SPB-NE insertion. Our data illuminate cellular requirements for remodeling the NE during “closed” nuclear division and provide insight into the structure and functions of the eukaryotic TMEM33 family.     

Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution

The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope–NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.     

Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry

Disassembly of nuclear pore complexes (NPCs) is a decisive event during mitotic entry in cells undergoing open mitosis, yet the molecular mechanisms underlying NPC disassembly are unknown. Using chemical inhibition and depletion experiments we show that NPC disassembly is a phosphorylation-driven process, dependent on CDK1 activity and supported by members of the NIMA-related kinase (Nek) family. We identify phosphorylation of the GLFG-repeat nucleoporin Nup98 as an important step in mitotic NPC disassembly. Mitotic hyperphosphorylation of Nup98 is accomplished by multiple kinases, including CDK1 and Neks. Nuclei carrying a phosphodeficient mutant of Nup98 undergo nuclear envelope breakdown slowly, such that both the dissociation of Nup98 from NPCs and the permeabilization of the nuclear envelope are delayed. Together, our data provide evidence for a phosphorylation-dependent mechanism underlying disintegration of NPCs during prophase. Moreover, we identify mitotic phosphorylation of Nup98 as a rate-limiting step in mitotic NPC disassembly.     

Spatial and temporal control of nuclear envelope assembly by Ran GTPase

Using evidence derived primarily from studies using Xenopus egg extracts, a model for the role of Ran in multiple stages during NE assembly can be proposed (Figure 2). Ran is concentrated on chromatin prior to NE assembly and recruits RCC1 that generates Ran-GTP locally. Recruitment of RCC1 to chromatin may be a specialized mechanism to initiate NE assembly following fertilization of the egg, whereas in somatic cells, RCC1 may be present on chromatin throughout mitosis. Ran-GTP recruits vesicles to the surface of chromatin, and promotes vesicle fusion to form the double membrane of the NE. Ran-GTP may recruit membrane vesicles to chromatin through binding to integral membrane proteins through importin-beta. A transient complex would be formed between Ran-GTP, importin-beta and the target protein, which would be released locally to promote assembly of a precursor complex. GTP hydrolysis by Ran would release importin-beta, but may also play a role in vesicle fusion. Ran-GTP also promotes NPC assembly by releasing nucleoporins such as Nup107 from inhibitory complexes with importin-beta. In vertebrate cells undergoing mitosis, the majority of Ran molecules are excluded from the chromosomes and dispersed into the cytoplasm. Relocalization of Ran to chromatin at the end of mitosis may co-ordinate the initiation of NE assembly with disassembly of the mitotic spindle. The function of Ran in this transition is likely to be coupled to changes in the activity of cyclin-dependent protein kinases and other activities that control the progression of the cell cycle. Thus, changes in the localization of Ran and its regulators provide temporal and spatial control of NE assembly at the end of mitosis.     

The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in the nuclear envelope (NE), through which exchange of molecules between the nucleus and cytosol occurs. Biogenesis of NPCs is complex and poorly understood. In particular, almost nothing is known about how NPCs are anchored in the NE. Here, we characterize vertebrate NDC1--a transmembrane nucleoporin conserved between yeast and metazoans. We show by RNA interference (RNAi) and biochemical depletion that NDC1 plays an important role in NPC and NE assembly in vivo and in vitro. RNAi experiments suggest a functional link between NDC1 and the soluble nucleoporins Nup93, Nup53, and Nup205. Importantly, NDC1 interacts with Nup53 in vitro. This suggests that NDC1 function involves forming a link between the NE membrane and soluble nucleoporins, thereby anchoring the NPC in the membrane.     

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1''s role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.     

Werner complex deficiency in cells disrupts the Nuclear Pore Complex and the distribution of lamin B1

From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of “RAN holes” void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus.     

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.     

Accelerating the rate of disassembly of karyopherin.cargo complexes

Transport of macromolecules across the nuclear pore complex (NPC) occurs in seconds and involves assembly of a karyopherin.cargo complex and docking to the NPC, translocation of the complex across the NPC via interaction with nucleoporins (Nups), and dissociation of the complex in the nucleoplasm. To identify rate-limiting steps in the Kap95p.Kap60p-mediated nuclear import pathway of Saccharomyces cerevisiae, we reconstituted key intermediate complexes and measured their rates of dissociation and affinities of interaction. We found that a nuclear localization signal-containing protein (NLS-cargo) dissociates slowly from Kap60p monomers and Kap60p.Kap95p heterodimers with half-lives (t(12)) of 7 and 73 min, respectively; that Kap60p and Kap60p.NLS-cargo complexes dissociate slowly from Kap95p (t(12) = 36 and 73 min, respectively); and that Kap95p.Kap60p.NLS-cargo complexes and Kap95p.Kap60p heterodimers dissociate rapidly from the nucleoporin Nup1p (t(12) < or = 21 s) and other Nups. A search for factors that accelerate disassembly of the long-lived intermediates revealed that Nup1p and Nup2p accelerate 16- and 19-fold the rate of dissociation of NLS-cargo from Kap60p.Kap95p heterodimers; that Gsp1p-GTP accelerates > or = 447-fold the rate of dissociation of Kap60p.NLS-cargo from Kap95p; and that Nup2p and the Cse1p.Gsp1p-GTP complex independently accelerate > or = 22- and > or = 39-fold the rate of dissociation of NLS-cargo from Kap60p. We suggest that Nup1p, Nup2p, Cse1p, and Gsp1p accelerate disassembly of Kap95p.Kap60p.NLS-cargo complexes by triggering allosteric mechanisms within Kaps that cause rapid release of binding partners. In that way, Nup1p, Nup2p, Cse1p, and Gsp1p may function as karyopherin release factors (or KaRFs) in the nuclear basket structure of the S. cerevisiae NPC.     

Microtubule-associated nuclear envelope proteins in interphase and mitosis

The LINC (linker of nucleoskeleton and cytoskeleton) complex forms a transcisternal bridge across the NE (nuclear envelope) that connects the cytoskeleton with the nuclear interior. This enables some proteins of the NE to communicate with the centrosome and the microtubule cytoskeleton. The position of the centrosome relative to the NE is of vital importance for many cell functions, such as cell migration and division, and centrosomal dislocation is a frequent phenotype in laminopathic disorders. Also in mitosis, a small group of transmembrane NE proteins associate with microtubules when they concentrate in a specific membrane domain associated with the mitotic spindle. The present review discusses structural and functional aspects of microtubule association with NE proteins and how this association may be maintained over the cell cycle.