Metastable network model of protein transport through nuclear pores

To reconcile the observed selectivity and the high rate of translocation of cargo-importin complexes through nuclear pores, we propose that the core of the nuclear pore complex is blocked by a metastable network of phenylalanine and glycine nucleoporins. Although the network arrests the unfacilitated passage of objects larger than its mesh size, cargo-importin complexes act as catalysts that reduce the free energy barrier between the cross-linked and the dissociated states of the Nups, and open the network. Using Brownian dynamics simulations we calculate the distribution of passage times through the network for inert particles and cargo-importin complexes of different sizes and discuss the implications of our results for experiments on translocation of proteins through the nuclear pore complex.     

Octagonal nuclear pores

Negative staining of isolated nuclear envelopes by phosphotungstate shows that the nuclear pores are octagonal rather than circular. Pores of the same shape and approximately the same width, 663 ± 5 A, were demonstrated in the newt, Triturus, the frog, Rana, and the starfish, Henricia. The outer and inner diameters of the annulus associated with each pore are respectively greater and less than the width of the pore itself. For this reason surface views of the envelope, unless negatively stained, fail to show the true dimensions of the pores.     

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

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Movement of a karyophilic protein through the nuclear pores of oocytes

It has recently been shown that large karyophilic proteins are transported across the nuclear envelope in amphibian oocytes. In consideration of this, the present experiments were performed to identify the specific sites within the envelope through which transport occurs and determine if molecular size is a limiting factor in the transport process. The following experimental procedure was employed: Colloidal gold particles, varying in size from approximately 20 to 170 A in diameter were coated with nucleoplasmin, a 165,000-mol-wt karyophilic protein, which is known to be transported through the envelope. The coated gold particles were microinjected into the cytoplasm of Xenopus oocytes, and the cells were fixed 15 min and 1 h later. The intracellular localization of the gold was then determined with the electron microscope. It was found that nucleoplasmin-coated particles readily enter the nucleus. On the basis of the distribution of the particles associated with the envelope, we concluded that transport occurs through the nuclear pores. Furthermore, the size distributions of the gold particles present in the nucleus and cytoplasm were not significantly different, indicating that the envelope does not discriminate among particles with diameters ranging from 50 to 200 A (the dimensions including the nucleoplasmin coat). Colloidal gold coated with trypsin-digested nucleoplasmin (which lacks the polypeptide domain required for transport) or exogenous polyvinylpyrrolidone were largely excluded from the nucleus and showed no evidence of transport.     

Translocation of RNA-coated gold particles through the nuclear pores of oocytes

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20-230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.     

Structural organization, functions and dynamics of nuclear pores

This review summarized data on the morphological and biochemical analysis of nuclear pore complexes, which are complex organelles providing the route of passive and active nuclear-cytoplasmic transport to different molecules in the eukaryotic cell. The morphology and functional role of nuclear pores in higher and lower eukaryotes, and molecular aspects of the import and export of molecules from the nucleus are described in addition to factors involved in the regulation of these process. Special attention has been paid to sequential steps of the nuclear pore assembly in vitro and in vivo.     

The subunit structure of chromatin fibres

Optimal conditions for studying the ultrastructure of chromatin fibers of histone-containing spermatozoa in thin sections have been determined. Better results for preservation in sperm of the sea cucumber Holothuria tubulosa, have been found than in different frog species studied. The fine structure of chromatin fibers after different treatments was studied by computer methods. A clear superbead structure was found under all conditions which preserve the chromatin fibres. These have a diameter of 30 nm, with superbeads about 33 nm long. In the best preserved cases an additional periodicity of 11 nm along the fibres was found. There is no clear relationship of this periodicity with an eventual solenoidal structure of the chromatin fibers.     

Permeability of single nuclear pores.

In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tsch?drich-Rotter and Peters. 1998. J. Microsc. 192:114-125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of approximately 4, 10, and 20 kDa. Dextrans of >/=40 kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4-6.1 nm (mean 5. 35 nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40-50 nm (mean 44.5 nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress.     

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.     

Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.