Influence of cargo size on Ran and energy requirements for nuclear protein import (original) (raw)
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Molecular and cellular biology, 1999
Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family. After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel. Once cargo is released, the importin then moves back through the channel for new rounds of transport. Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm. In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP). Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies. Pse1-GFP was studied in the most detail. Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that ...
Journal of Cell Biology, 2002
he nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cyto-T plasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin ␣ /  -or transportin-dependent import.
Dynamic localization of the nuclear import receptor and its interactions with transport factors
The Journal of Cell Biology, 1996
Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rnal-1 strain, we have identified a genetic interaction between Rnalp, a GTPase activating protein required for nuclear transport, and yeast importin-[3, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rnalp physically interacts with importin-~. Mutants in importin-~ exhibit in vivo nuclear protein import defects, and importin-[3 localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-ot, but not importin-13, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-a and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-a from the nucleus.
The Molecular Mechanism of Transport of Macromolecules Through Nuclear Pore Complexes
Traffic, 2000
Trafficking of macromolecules between nuclear and cytoplasmic compartments takes place through the nuclear pore complexes (NPCs) of the nuclear envelope. Nuclear trafficking involves a complex series of interactions between cargo, soluble transport factors (carriers) and nuclear pore proteins (nucleoporins) that are orchestrated by the Ras-family GTPase Ran. The primary role of Ran is probably to establish directionality and to sort molecules to be transported by controlling the interaction between carriers and cargoes, so that they bind in one compartment but dissociate in the other. Translocation of carriers and cargo-carrier complexes through NPCs requires interactions between the carriers and nucleoporins that contain distinctive tandem sequence repeats based on cores rich in glycine and phenylalanine residues that are separated by hydrophilic linkers. Much recent work has focused on these interactions and, in particular, their specificity, regulation and function. Evidence is accumulating that carriers move through the NPC by distinct but overlapping routes using specific subsets of nucleoporins.
The Mechanism of Nucleocytoplasmic Transport through the Nuclear Pore Complex
Cold Spring Harbor Symposia on Quantitative Biology, 2010
Unlike their prokaryotic ancestors, eukaryotic cells contain numerous membrane-bound organelles that compartmentalize various specialized processes. This evolutionary advancement enabled tighter regulation of cellular functions but meant that cells needed to develop channels to transport proteins, ions, and other substances specifically across their internal and external membranes. Of all of the transport channels in a cell, one stands out from all of the others in terms of the range and size of cargoes that pass through it. This is the NPC, which mediates all transport across the nuclear envelope (NE) (the double-membraned layer that separates the nuclear genetic material from the cytoplasm). Transport through the NPC is rapid and bidirectional, with a large set of diverse cargoes crossing between the nucleus and cytoplasm. Cargoes such as transcription factors, ribosomal proteins, and viral nucleic acids enter the nucleus, whereas, messenger ribonucleoproteins (mRNPs), transfer RNAs (tRNAs), and ribosomal subunits exit. Despite the wide range of molecules that must pass through the NPC, nucleocytoplasmic transport is incredibly selective. Transport can also occur very quickly, up to the order of thousands of events per NPC per second (Ribbeck and Gorlich 2001). Yet the structure of the NPC, discussed below, has been found to be surprisingly simple. How this simple structure might mediate this rapid yet selective transport of such a variety of cargoes remains the main question in the field and is the major topic of this chapter. SOLUBLE PHASE OF TRANSPORT Karyopherin-Mediated Transport Numerous lines of evidence have shown that only proteins that can specifically bind to the family of nuclear pore proteins called FG nups are able to efficiently and rapidly cross the NPC. Many such FG-binding proteins act as transport factors, carrying non-FG-binding cargoes across the NPC. Karyopherins (Kaps), also known as importins and exportins, are a family of transport factors that carry proteins and protein/RNA complexes as large as 2 MDa across the NPC (Dworetzky and Feldherr 1988;
Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex
Traffic, 2013
Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG-Nups in the central channel, and the linker can position the transport factor-bound NLS in the vicinity of the FG-Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin-mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC.
Journal of Molecular Biology, 1997
Nuclear transport factor 2 (NTF2) is associated with the translocation stage of nuclear protein import and binds both to nuclear pore proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2±Ran interaction in nuclear protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule's hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Puri®ed mutant proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant protein showed signi®cant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell nuclear protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for ef®cient nuclear protein import.
The Importin β Binding Domain Modulates the Avidity of Importin β for the Nuclear Pore Complex
Journal of Biological Chemistry, 2010
Importin  mediates active passage of cellular substrates through the nuclear pore complex (NPC). Adaptors such as importin ␣ and snurportin associate with importin  via an importin  binding (IBB) domain. The intrinsic structural flexibility of importin  allows its concerted interactions with IBB domains, phenylalanine-glycine nucleoporins, and the GTPase Ran during transport. In this paper, we provide evidence that the nature of the IBB domain modulates the affinity of the import complex for the NPC. In permeabilized cells, importin  imports a cargo fused to the snurportin IBB (sIBB) with ϳ70% reduced energy requirement as compared with the classical importin ␣ IBB. At the molecular level, this is explained by ϳ200-fold reduced affinity of importin  for Nup62, when bound to the sIBB. Consistently, in vivo, the importin ⅐sIBB complex has greatly reduced persistence inside the central channel of the NPC. We propose that by controlling the degree of strain in the tertiary structure of importin , the IBB domain modulates the affinity of the import complex for nucleoporins, thus dictating its persistence inside the NPC.