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1.
While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future. 相似文献
2.
Xunbin Wei Vanessa G. Henke Carsten Strübing Edward B. Brown David E. Clapham 《Biophysical journal》2003,84(2):1317-1327
The NPC is the portal for the exchange of proteins, mRNA, and ions between nucleus and cytoplasm. Many small molecules (<10 kDa) permeate the nucleus by simple diffusion through the pore, but molecules larger than 70 kDa require ATP and a nuclear localization sequence for their transport. In isolated Xenopus oocyte nuclei, diffusion of intermediate-sized molecules appears to be regulated by the NPC, dependent upon [Ca2+] in the nuclear envelope. We have applied real-time imaging and fluorescence recovery after photobleaching to examine the nuclear pore permeability of 27-kDa EGFP in single intact cells. We found that EGFP diffused bidirectionally via the NPC across the nuclear envelope. Although diffusion is slowed ~100-fold at the nuclear envelope boundary compared to diffusion within the nucleus or cytoplasm, this delay is expected for the reduced cross-sectional area of the NPCs. We found no evidence for significant nuclear pore gating or block of EGFP diffusion by depletion of perinuclear Ca2+ stores, as assayed by a nuclear cisterna-targeted Ca2+ indicator. We also found that EGFP exchange was not altered significantly during the cell cycle. 相似文献
3.
Jens Popken Alexander Graf Stefan Krebs Helmut Blum Volker J. Schmid Axel Strauss Tuna Guengoer Valeri Zakhartchenko Eckhard Wolf Thomas Cremer 《PloS one》2015,10(5)
The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA. 相似文献
4.
A nuclear pore complex (NPC) is a large protein assembly that mediates the nucleocytoplasmic exchange of molecules. During the cell cycle, NPCs assemble, disassemble, and dynamically change their distribution on assembled nuclear envelope (NE), whereas in post-mitosis, NPCs are extremely stable. Extensive studies on its components, structure, and building blocks allow the study of its assembly and disassembly at the molecular level. Depending on the location that the initial components of this structure are built (e.g. chromatin versus double lipid bilayers of the nuclear envelope), the regulation and the mechanism of the assembly differ. Moreover, cell cycle dynamics of NPC are linked with INM proteins, lamins, lipid membranes, and the cell cycle signal, which show that NPC dynamics are highly regulated processes. 相似文献
5.
K. R. Straatman J. Nijsse H. Kieft A. C. van Aelst J. H. N. Schel 《Sexual plant reproduction》2000,13(1):43-51
Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm3, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed.
The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation
of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm2 at the stage of isolation to 9 NPCs/μm2, under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing
the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm2, whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm2. This low density of 9 NPCs/μm2 was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm2 found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically
rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei.
In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm2, indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper.
In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented.
Received: 29 December 1999 / Revision accepted: 3 March 2000 相似文献
6.
A novel allele of Saccharomyces cerevisiae NDC1 reveals a potential role for the spindle pole body component Ndc1p in nuclear pore assembly
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Both the spindle pole body (SPB) and the nuclear pore complex (NPC) are essential organelles embedded in the nuclear envelope throughout the life cycle of the budding yeast Saccharomyces cerevisiae. However, the mechanism by which these two multisubunit structures are inserted into the nuclear envelope during their biogenesis is not well understood. We have previously shown that Ndc1p is the only known integral membrane protein that localizes to both the SPBs and the NPCs and is required for SPB duplication. For this study, we generated a novel temperature-sensitive (ts) allele of NDC1 to investigate the role of Ndc1p at the NPCs. Yeast cells carrying this allele (ndc1-39) failed to insert the SPB into the nuclear envelope at the restrictive temperature. Importantly, the double mutation of ndc1-39 and NPC assembly mutant nic96-1 resulted in cells with enhanced growth defects. While nuclear protein import and NPC distribution in the nuclear envelope were unaffected, ndc1-39 mutants failed to properly incorporate the nucleoporin Nup49p into NPCs. These results provide evidence that Ndc1p is required for NPC assembly in addition to its role in SPB duplication. We postulate that Ndc1p is crucial for the biogenesis of both the SPBs and the NPCs at the step of insertion into the nuclear envelope. 相似文献
7.
Jindriska Fiserova Elena Kiseleva Martin W. Goldberg 《The Plant journal : for cell and molecular biology》2009,59(2):243-255
The nuclear envelope (NE) is a fundamental structure of eukaryotic cells with a dual role: it separates two distinct compartments, and enables communication between them via nuclear pore complexes (NPCs). Little is known about NPCs and NE structural organization in plants. We investigated the structure of NPCs from both sides of the NE in tobacco BY-2 cells. We detected structural differences between the NPCs of dividing and quiescent nuclei. Importantly, we also traced the organizational pattern of the NPCs, and observed non-random NPC distribution over the nuclear surface. Lastly, we observed an organized filamentous protein structure that underlies the inner nuclear membrane, and interconnects NPCs. The results are discussed within the context of the current understanding of NE structure and function in higher eukaryotes. 相似文献
8.
Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. 总被引:24,自引:0,他引:24
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N Daigle J Beaudouin L Hartnell G Imreh E Hallberg J Lippincott-Schwartz J Ellenberg 《The Journal of cell biology》2001,154(1):71-84
The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis. 相似文献
9.
Nuclear pore complexes (NPCs) serve as transport channels across the nuclear membrane, a double lipid bilayer that physically
separates the nucleoplasm and cytoplasm of eukaryotic cells. New evidence suggests that the multiprotein nuclear pores also
play a role in chromatin organization and gene expression. Given the importance of NPC function, it is not surprising that
a growing list of human diseases and developmental defects have been linked to its malfunction. In order to fully understand
the functional repertoire of NPCs and their essential role for nuclear organization, it is critical to determine the sequence
of events that lead to the formation of nuclear pores. This is particularly relevant since NPC number, and possibly composition,
are tightly linked to metabolic activity. Most of our knowledge is derived from NPC formation that occurs in dividing cells
at the end of mitosis when the nuclear envelope (NE) and NPCs reform from disassembled precursors. However, NPC assembly also
takes place during interphase into an intact NE. Importantly, this process is not restricted to dividing cells but also occurs
during cell differentiation. Here, we will review aspects unique to this process, namely the regulation of nuclear expansion
and the mechanisms of fusion between the outer and inner nuclear membranes. We will then discuss conserved and diverging mechanisms
between post-mitotic and interphase assembly of the proteinaceous structure in light of recently published data. 相似文献
10.
In vivo studies on the dynamics of the nuclear pore complex (NPC) in yeast suggested that NPCs are highly mobile in the nuclear envelope. However, new evidence indicates that in mammalian cells NPCs are stably attached to a flexible lamina framework, but a peripheral component can exchange rapidly with an intranuclear pool. 相似文献
11.
Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene 总被引:11,自引:2,他引:9
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《The Journal of cell biology》1995,131(6):1677-1697
To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7- 1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p. 相似文献
12.
13.
T. Danker V. Shahin A. Schlune C. Schäfer H. Oberleithner 《The Journal of membrane biology》2001,184(2):91-99
The nuclear hourglass technique (NHT) was recently introduced as a novel technique that measures the electrical nuclear envelope
(NE) conductance of isolated Xenopus laevis oocyte nuclei. The main conclusion drawn from NHT work so far is that nuclear pore complexes (NPCs) of oocytes are in an
electrically open state under physiological conditions, with a mean conductance of 1.7 nS per NPC. Since nuclear patch-clamp
data indicate that usually NPCs are electrically closed, our work has been challenged by the notion that NHT cannot assure
a high resistance seal (``gigaseal') between glass wall and NE like that required for patch-clamp experiments. Thus, NHT
could have dramatically underestimated NE electrical resistance. Here we demonstrate that NHT does not require a gigaseal
for accurate NE conductance measurements. In addition, we present experimental conditions where mean single NPC electrical
conductance is reduced 26-fold due to electrophoretic plugging by negatively charged nucleoplasmic macromolecules. In addition,
data indicate that under physiological conditions (i.e., when macromolecules are offered in the cytosolic solution) the nuclear
surface is heavily folded, underestimating ``true' NE surface by a factor of 2.6. When ``true' NE surface area is taken
into consideration, modified values of mean single NPC conductances of 654 pS for electrically open conditions and 25 pS for
electrically plugged conditions can be calculated.
We conclude that the large overall NE conductance detected with the nuclear hourglass technique in intact Xenopus laevis oocyte nuclei can be explained by the sum of single NPC conductances in the pS range, as long as open probability is high.
This confirms previous patch-clamp work concerning single NPC conductance, but disagrees with the view that mean open probability
of NPC channels is usually low.
Received: 27 March 2001/Revised: 3 July 2001 相似文献
14.
核孔复合物(NPC)是一个巨型分子复合物,相对分子质量约125×106。脊椎动物的NPC由大约30种蛋白质组成,这些蛋白质的序列大多具有FG(苯丙氨酸-甘氨酸)重复序列。NPC锚定于双层核膜上,并且是物质跨核膜运输的惟一通道,它可快速介导小分子物质的被动运输以及大分子物质的主动运输过程。虽然NPC具有较大的相对分子质量和复杂的结构,但它可在细胞分裂过程中分离并重新组装。生物大分子经NPC的跨核膜运输直接影响真核细胞的生长、增殖、分化、发育等多种生命活动。本文重点介绍NPC的结构、组装及其功能特点。 相似文献
15.
Kazuhiro Furukawa Kazuya Ishida Taka-aki Tsunoyama Shinichi Osoda Paul A. Fisher 《Experimental cell research》2009,315(7):1181-1090
To investigate nuclear lamina re-assembly in vivo, Drosophila A-type and B-type lamins were artificially expressed in Drosophila lamin Dm0null mutant brain cells. Both exogenous lamin C (A-type) and Dm0 (B-type) formed sub-layers at the nuclear periphery, and efficiently reverted the abnormal clustering of the NPC. Lamin C initially appeared where NPCs were clustered, and subsequently extended along the nuclear periphery accompanied by the recovery of the regular distribution of NPCs. In contrast, lamin Dm0 did not show association with the clustered NPCs during lamina formation and NPC spacing recovered only after completion of a closed lamin Dm0 layer. Further, when lamin Dm0 and C were both expressed, they did not co-polymerize, initiating layer formation in separate regions. Thus, A and B-type lamins reveal differing properties during lamina assembly, with A-type having the primary role in organizing NPC distribution. This previously unknown complexity in the assembly of the nuclear lamina could be the basis for intricate nuclear envelope functions. 相似文献
16.
Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures is the nucleus, which is bounded by a double membrane termed the nuclear envelope (NE). Though this NE separates the nucleoplasm and genetic material within the nucleus from the surrounding cytoplasm, it is studded throughout with portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of protein and ribonucleoprotein cargoes. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, and the NPC carries binding sites for many of the proteins that modulate and modify the cargoes as they pass across the NE. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation. All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.Taxonomically speaking, all life on earth falls into one of two fundamental groups, the prokaryotes and the eukaryotes. The prokaryotes, the first group to evolve, are single cell organisms bounded by a single membrane. About 1.5 billion years later, a series of evolutionary innovations led to the emergence of eukaryotes. Eukaryotes have multiple inner membrane structures that allow for compartmentalization within the cell, and therefore differentiation of the cell and regulation within it. Ultimately, the greater cellular complexity of eukaryotes allowed them to adopt a multicellular lifestyle, as seen in the plants, fungi and animals of today (reviewed in Field and Dacks 2009).Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures, which gives the eukaryotes their Greek-rooted name, is the nucleus—the central “kernel” (gr. “karyo-”) of the cell. The nucleus is bounded by a double membrane termed the nuclear envelope (NE), which separates the nucleoplasm and genetic material from the surrounding cytoplasm. However the genetic material in the nucleus is not totally isolated from the rest of the cell. Studded throughout the NE are portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of cargoes. Going into the nucleus, these cargoes include inner nuclear membrane proteins and all the proteins in the nucleoplasm. Going out are RNA-associated proteins that are assembled into ribosomal subunits or messenger ribonucleoproteins (mRNPs). Once transported, the NPC must ensure these cargos are retained in their respective nuclear and cytoplasmic compartments. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, providing a powerful extra level of cellular control that is not necessary in prokaryotes. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation (reviewed in Ahmed and Brickner 2007; Hetzer and Wente 2009). All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes. 相似文献
17.
The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin. 相似文献
18.
Mark Kittisopikul Takeshi Shimi Meltem Tatli Joseph Riley Tran Yixian Zheng Ohad Medalia Khuloud Jaqaman Stephen A. Adam Robert D. Goldman 《The Journal of cell biology》2021,220(4)
Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D–structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna−/− and Lmnb1−/− fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope. 相似文献
19.
Nuclear pore complex assembly through the cell cycle: regulation and membrane organization 总被引:1,自引:0,他引:1
In eukaryotes, all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), which are among the largest supramolecular assemblies in cells. Although their composition in yeast and metazoa is well characterized, understanding how NPCs are assembled and form the pore through the double membrane of the nuclear envelope and how both processes are controlled still remains a challenge. Here, we summarize what is known about the biogenesis of NPCs throughout the cell cycle with special focus on the membrane reorganization and the regulation that go along with NPC assembly. 相似文献
20.
A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly 总被引:1,自引:0,他引:1
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Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis. 相似文献