Kinetic analysis of translocation through nuclear pore complexes

The mechanism of facilitated translocation through nuclear pore complexes (NPCs) is only poorly understood. Here, we present a kinetic analysis of the process using various model substrates. We find that the translocation capacity of NPCs is unexpectedly high, with a single NPC allowing a mass flow of nearly 100 MDa/s and rates in the order of 103 translocation events per second. Our data further indicate that high affinity interactions between the translocation substrate and NPC components are dispensable for translocation. We propose a 'selective phase model’ that could explain how NPCs function as a permeability barrier for inert molecules and yet become selectively permeable for nuclear transport receptors and receptor–cargo complexes.

[1]  L. C. Moore,et al.  Nuclear envelope permeability , 1975, Nature.

[2]  U. Kubitscheck,et al.  Single nuclear pores visualized by confocal microscopy and image processing. , 1996, Biophysical journal.

[3]  J. Kuhlmann,et al.  Dissecting the Interactions between NTF2, RanGDP, and the Nucleoporin XFXFG Repeats* , 2000, The Journal of Biological Chemistry.

[4]  C. Akey,et al.  Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. , 1998, Molecular cell.

[5]  G. Dreyfuss,et al.  Transport of Proteins and RNAs in and out of the Nucleus , 1999, Cell.

[6]  U. Kutay,et al.  The translocation of transportin–cargo complexes through nuclear pores is independent of both Ran and energy , 1999, Current Biology.

[7]  F. Bischoff,et al.  Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1 , 1991, Nature.

[8]  G. Dreyfuss,et al.  Import and export of the nuclear protein import receptor transportin by a mechanism independent of GTP hydrolysis , 1998, Current Biology.

[9]  J. Olivo,et al.  Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis , 1999, Current Biology.

[10]  M. Canela,et al.  Accuracy and precision in the determination of Stokes radii and molecular masses of proteins by gel filtration chromatography. , 1989, Journal of Chromatography A.

[11]  Dirk Görlich,et al.  RanBP1 is crucial for the release of RanGTP from importin β‐related nuclear transport factors , 1997, FEBS letters.

[12]  I. Mattaj,et al.  Nucleocytoplasmic transport: the soluble phase. , 1998, Annual review of biochemistry.

[13]  K. Weis,et al.  Importins and exportins: how to get in and out of the nucleus. , 1998, Trends in biochemical sciences.

[14]  E. Buhle,et al.  Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components , 1990, The Journal of cell biology.

[15]  J Langowski,et al.  Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy. , 2000, Journal of molecular biology.

[16]  U. Kutay,et al.  Transport between the cell nucleus and the cytoplasm. , 1999, Annual review of cell and developmental biology.

[17]  B. Chait,et al.  The Yeast Nuclear Pore Complex: Composition, Architecture, and Transport Mechanism , 2000 .

[18]  I. Macara,et al.  Nuclear Import of the Ran Exchange Factor, Rcc1, Is Mediated by at Least Two Distinct Mechanisms , 2000, The Journal of cell biology.

[19]  E. Schwoebel,et al.  Ran-dependent Signal-mediated Nuclear Import Does Not Require GTP Hydrolysis by Ran* , 1998, The Journal of Biological Chemistry.

[20]  G. Blobel,et al.  The GTP-binding protein Ran/TC4 is required for protein import into the nucleus , 1993, Nature.

[21]  E. Hurt,et al.  Yeast genetics to dissect the nuclear pore complex and nucleocytoplasmic trafficking. , 1997, Annual review of genetics.

[22]  G. Blobel,et al.  Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins , 1995, Cell.

[23]  A. McCoy,et al.  Structural basis for molecular recognition between nuclear transport factor 2 (NTF2) and the GDP-bound form of the Ras-family GTPase Ran. , 1998, Journal of molecular biology.

[24]  K. Zerf,et al.  Optical recording of signal-mediated protein transport through single nuclear pore complexes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[25]  G. Stein,et al.  TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE , 1972, The Journal of cell biology.

[26]  G. Blobel,et al.  Purification of a Ran-interacting protein that is required for protein import into the nucleus. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[27]  F. Bischoff,et al.  Dominant‐negative mutants of importin‐β block multiple pathways of import and export through the nuclear pore complex , 1997, The EMBO journal.

[28]  I. Macara,et al.  Nuclear import of Ran is mediated by the transport factor NTF2 , 1998, Current Biology.

[29]  Ed Hurt,et al.  Binding of the Mex67p/Mtr2p Heterodimer to Fxfg, Glfg, and Fg Repeat Nucleoporins Is Essential for Nuclear mRNA Export , 2000, The Journal of cell biology.

[30]  M. Rout,et al.  Isolation of the yeast nuclear pore complex , 1993, The Journal of cell biology.

[31]  S. Adam,et al.  Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors , 1990, The Journal of cell biology.

[32]  G. Dreyfuss,et al.  A Novel Receptor-Mediated Nuclear Protein Import Pathway , 1996, Cell.

[33]  B. Paschal,et al.  Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62 , 1995, The Journal of cell biology.

[34]  P. Silver,et al.  Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex. , 2000, Molecular cell.

[35]  G. Lipowsky,et al.  NTF2 mediates nuclear import of Ran , 1998, The EMBO journal.

[36]  C. Feldherr,et al.  Movement of a karyophilic protein through the nuclear pores of oocytes , 1984, The Journal of cell biology.

[37]  F. Bischoff,et al.  Export of Importin α from the Nucleus Is Mediated by a Specific Nuclear Transport Factor , 1997, Cell.

[38]  C. Feldherr.  THE NUCLEAR ANNULI AS PATHWAYS FOR NUCLEOCYTOPLASMIC EXCHANGES , 1962, The Journal of cell biology.

[39]  Richard Bayliss,et al.  Structural Basis for the Interaction between FxFG Nucleoporin Repeats and Importin-β in Nuclear Trafficking , 2000, Cell.

[40]  F. Bischoff,et al.  The importin β/importin 7 heterodimer is a functional nuclear import receptor for histone H1 , 1999, The EMBO journal.

[41]  F. Bischoff,et al.  Identification of different roles for RanGDP and RanGTP in nuclear protein import. , 1996, The EMBO journal.

[42]  H. Busch.  The Cell Nucleus , 1966, Nature.

[43]  D. Görlich,et al.  Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP. , 1999, Journal of molecular biology.

[44]  S. R. Wente,et al.  The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. , 2000, Current opinion in cell biology.

[45]  M. Radermacher,et al.  Architecture of the Xenopus nuclear pore complex revealed by three- dimensional cryo-electron microscopy , 1993, The Journal of cell biology.

[46]  G. Blobel,et al.  The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex , 1995, Cell.

[47]  U. Kutay,et al.  The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. , 2000, RNA.

[48]  D. Goldfarb,et al.  Protein import through the nuclear pore complex is a multistep process , 1989, The Journal of cell biology.

[49]  M. Stewart,et al.  Separate binding sites on nuclear transport factor 2 (NTF2) for GDP-Ran and the phenylalanine-rich repeat regions of nucleoporins p62 and Nsp1p. , 1996, Journal of molecular biology.

[50]  N. Imamoto,et al.  Ran-unassisted Nuclear Migration of a 97-kD Component of Nuclear Pore–targeting Complex , 1997, The Journal of cell biology.