Passive and Facilitated Transport in Nuclear Pore Complexes Is Largely Uncoupled

Abstract

Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.Eukaryotic cell nuclei are separated from the cytoplasm by a double lipid bilayer system known as the nuclear envelope (NE).2 Exchange of material between the two compartments proceeds through nuclear pore complexes (NPCs), large protein assemblies that span the NE and provide the sole medium for exchange. The vertebrate NPC has a molecular mass of ϳ125 MDa (1) and is made up of ϳ30 different proteins, called nucleoporins, most of which are present in multiples of eight (2, 3). The core of the NPC consists of a symmetrical framework, measuring ϳ120 ϫ 90 nm, which is made of two coaxial rings sandwiching a wheel-like array of eight spoke-shaped domains. The spoke-ring assembly encircles the central pore channel, which resembles an hourglass 45-50 nm wide at its waist (4 -7). In addition to the central framework, NPCs contain peripheral structures, which are anchored to the ring moieties of the spoke-ring assembly and serve as docking sites for nuclear transport receptors and effectors. These structures include eight short (ϳ50 nm) filaments that protrude toward the cytoplasm and a massive, fish trap-like structure, termed the nuclear basket, which extends into the nucleus (4 -11). Yeast NPCs have an overall similar architecture but are smaller (3,(12)(13)(14).Transport across the NPC has been reviewed in detail (15-22) and can be divided into two modes. Small molecules, such as ions, metabolites, and intermediate-sized macromolecules, can pass unassisted by diffusion which becomes increasingly restricted as the particle approaches a size limit of ϳ10 nm in diameter (23,24). In contrast, proteins, RNAs, and their complexes are ushered selectively by dedicated soluble transport receptors, which recognize specific import (NLS) or nuclear export signal (NES) peptides displayed by the cargo. In most cases, assembly and disassembly of transport complexes in the appropriate cellular compartment are coordinated by the small GTPase Ran, which performs these activities by binding to transport receptors in its GTP-bound form. Facilitated translocation is often coupled to an input of metabolic energy that permits transport ...

Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport

“…indicates that the NE permeability to passive diffusion of NES-GFP is independent from NES-GFP or NLS-Cherry concentration. This implies that active export and passive transport are negligibly coupled in cells, consistently with the analogous behavior described for NLS-cargoes (19). Notably, the average value P NES ϭ 9.5 Ϯ 5 m 3 /s is comparable to the average permeability coefficient obtained for GFP alone (P GFP ϭ 13 Ϯ 4 m 3 /s) and NLS-GFP, P NLS ϭ 5.4 Ϯ 3 m 3 /s).…”

“…We shall show that our data highlight the similarity between export and fluxes across the NPC, and their complete uncoupling. Also, we find that passive diffusion does not interfere with nuclear export, analogous to that reported by Naim et al for nuclear import (19). We believe that these results can provide a useful reference framework for a thorough description of NPC.…”

“…2c). This uncoupling of the translocation mechanisms is strongly suggestive of a structural separation of these processes within the NPC, as recently proposed by others (18,19). Alternatively, in fact, one would have to require the fine tuning of the thermodynamic characteristics of active and passive fluxes inside the same NPC channel to prevent interference and allow their separate modulation.…”

“…A recent report based on immunoelectron microscopy indicated that energy-dependent transport (import and export) and diffusion may take distinct spatial routes through the NPC (18). Similarly, Naim et al (19) proposed that energy-dependent import and passive diffusion are not dynamically coupled.…”

Distinct, but not completely separate spatial transport routes in the nuclear pore complex

“…[43][44][45] Later, similar studies further indicated that these two transport modes were functionally independent, without any competition occurring between them. 46,47 However, at the same time, many other researchers argued against the above conclusions because they observed common pathways shared by passive and facilitated transport, not only spatially but also in terms of function. [48][49][50][51] The above disputes are reflected in the proposed transport models.…”