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Specific nucleoporins and nuclear pore complex–dependent transport events directly influence aging in yeast., The eukaryotic nuclear permeability barrier and selective nucleocytoplasmic transport are maintained by nuclear pore complexes (NPCs), large structures composed of ∼30 proteins (nucleoporins [Nups]). NPC structure and function are disrupted in aged nondividing metazoan cells, although it is unclear whether these changes are a cause or consequence of aging. Using the replicative life span (RLS) of Saccharomyces cerevisiae as a model, we find that specific Nups and transport events regulate longevity independent of changes in NPC permeability. Mutants lacking the GLFG domain of Nup116 displayed decreased RLSs, whereas longevity was increased in nup100-null mutants. We show that Nup116 mediates nuclear import of the karyopherin Kap121, and each protein is required for mitochondrial function. Both Kap121-dependent transport and Nup116 levels decrease in replicatively aged yeast. Overexpression of GSP1, the small GTPase that powers karyopherin-mediated transport, rescued mitochondrial and RLS defects in nup116 mutants and increased longevity in wild-type cells. Together, these studies reveal that specific NPC nuclear transport events directly influence aging.

Passive macromolecular diffusion through nuclear pore complexes is thought to decrease dramatically beyond ∼40 kD. Using time-resolved fluorescence microscopy and Brownian dynamics simulations, Timney et al. show that this barrier is in fact much softer, decreasing along a continuum., Passive macromolecular diffusion through nuclear pore complexes (NPCs) is thought to decrease dramatically beyond a 30–60-kD size threshold. Using thousands of independent time-resolved fluorescence microscopy measurements in vivo, we show that the NPC lacks such a firm size threshold; instead, it forms a soft barrier to passive diffusion that intensifies gradually with increasing molecular mass in both the wild-type and mutant strains with various subsets of phenylalanine-glycine (FG) domains and different levels of baseline passive permeability. Brownian dynamics simulations replicate these findings and indicate that the soft barrier results from the highly dynamic FG repeat domains and the diffusing macromolecules mutually constraining and competing for available volume in the interior of the NPC, setting up entropic repulsion forces. We found that FG domains with exceptionally high net charge and low hydropathy near the cytoplasmic end of the central channel contribute more strongly to obstruction of passive diffusion than to facilitated transport, revealing a compartmentalized functional arrangement within the NPC.

The yeast Saccharomyces cerevisiae is a common model organism for biological discovery. It has become popularized primarily because it is biochemically and genetically amenable for many fundamental studies on eukaryotic cells. These features, as well as the development of a number of procedures and reagents for isolating protein complexes, and for following macromolecules in vivo, have also fueled studies on nucleo-cytoplasmic transport in yeast. One limitation of using yeast to study transport has been the absence of a reconstituted in vitro system that yields quantitative data. However, advances in microscopy and data analysis have recently enabled quantitative nuclear import studies, which, when coupled with the significant advantages of yeast, promise to yield new fundamental insights into the mechanisms of nucleo-cytoplasmic transport.

Many cargoes destined for nuclear import carry nuclear localization signals that are recognized by karyopherins (Kaps). We present methods to quantitate import rates and measure Kap and cargo concentrations in single yeast cells in vivo, providing new insights into import kinetics. By systematically manipulating the amounts, types, and affinities of Kaps and cargos, we show that import rates in vivo are simply governed by the concentrations of Kaps and their cargo and the affinity between them. These rates fit to a straightforward pump–leak model for the import process. Unexpectedly, we deduced that the main limiting factor for import is the poor ability of Kaps and cargos to find each other in the cytoplasm in a background of overwhelming nonspecific competition, rather than other more obvious candidates such as the nuclear pore complex and Ran. It is likely that most of every import round is taken up by Kaps and nuclear localization signals sampling other cytoplasmic proteins as they locate each other in the cytoplasm.

Macromolecular traffic through the nuclear pore complex (NPC) occurs by either passive or facilitated diffusion. While passive diffusion rates are believed to decrease dramatically beyond the approximately 30-60 kDa size threshold, facilitated diffusion, which is mediated by soluble nuclear transport receptors (NTRs), proceeds rapidly even for large macromolecular assemblies such as intact ribosomal subunits. To study the mechanism by which the NPC facilitates rapid facilitated diffusion while maintaining an effective barrier to the passive diffusion of large macromolecules, we created a model of transport dynamics that integrates data from multiple empirical and theoretical sources: small-angle x-ray scattering, nuclear magnetic resonance spectroscopy, electron tomography, dynamic light scattering, atomistic simulations, and additional datasets available from the literature. In both our model and in independent fluorescence recovery after photobleaching (FRAP) measurements, we show that the barrier to passive diffusion is much softer than previously thought, and completely lacks a well-defined size cutoff at 30-60 kDa. Coarse-grained Brownian Dynamics simulations replicate these results, and indicate that this gradual decrease in passive diffusion rates with molecular weight is consistent with an entropic gating mechanism, in which repulsive forces are set out by the crowded and highly dynamic FG repeat domains that fill the central channel of the NPC. In facilitated diffusion, we show that cargo-bound NTRs counter these repulsive forces by utilizing a unique dynamic interaction mechanism with the FG repeats, transitioning between weakly- and strongly-interacting states, thereby sliding and exchanging between proximal FG repeats. This ‘slide-and-exchange’ interaction mechanism is reminiscent of the sliding of transcription factors on DNA molecules. Our results highlight the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolve the apparent conflict between the selectivity and speed of transport.

How do you build a sorting machine that can function with less than half of its sorting components? This problem challenges our understanding of nucleocytoplasmic transport after the finding that yeast can survive with nuclear pore complexes (NPCs) containing only half their original mass of transport-factor-binding domains.

In yeast there are at least 14 members of the beta-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast beta-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.

Summary The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC's inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2–960 [ScNup192(2–960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2–960) could undergo long-range transition between "open" and "closed" conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2–960), and homology modeling. Evolutionary analyses using the ScNup192(2–960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.