Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.DOI: http://dx.doi.org/10.7554/eLife.04052.001

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal ␤-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines.unfolding pathway ͉ transition state structure ͉ mechanical unfolding F olded proteins are Ϸ5-10 kcal⅐mol Ϫ1 more stable than their unfolded states. Therefore, the disassembly and translocation of folded proteins often require a molecular machine and a source of free energy. These ubiquitous multiprotein complexes include soluble degradation machinery, such as the proteasome or the Clp bacterial proteases (1), which unfold and degrade proteins, and some, but not all, membrane-embedded translocase channels, which can unfold and transport proteins across membranes (2). There are general features shared between these soluble and membrane-embedded translocase machines: a narrow central pore first engages the protein substrate on its free end, the substrate is unfolded mechanically, and the unfolded chain is translocated through the narrow pore, allowing it ultimately to either cross a membrane or enter into a proteolytic complex for degradation. Protein unfolding and translocation in these systems are often driven by ATP hydrolysis (1, 2), a membrane potential (⌬⌿) (2, 3), and/or a proton gradient (⌬pH) (4). The molecular mechanism of translocation-coupled unfolding, however, is poorly understood.Prior studies examining the correlation between substrate protein stability and translocation kinetics have produced conflicting results. Some ligand-stabilized substrates translocate inefficiently, because they are too thermodynamically stable (5); however, other substrates show little change in the rate of translocation when destabilized by mutagenesis (6, 7). To resolve these conflicting results, it was proposed that translocation-coupled unfolding (6, 8) depends on the mechanical stability of the local structure adjacent to the signal tag. O...

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