Reconstitution of Nuclear Import in Permeabilized Cells

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2008 Oct 28

PMID 18951186

Citations 29

Authors

Aurelia Cassany

Larry Gerace

Affiliations

Soon will be listed here.

Abstract

The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin-cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin alpha, importin beta, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.

Citing Articles

Toxoplasma Gondii Importin α Shows Weak Auto-Inhibition.

Bhambid M, Dey V, Walunj S, Patankar S Protein J. 2023; 42(4):327-342.

PMID: 37284905 DOI: 10.1007/s10930-023-10128-2.

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export.

Duan L, Zaepfel B, Aksenova V, Dasso M, Rothstein J, Kalab P Cell Rep. 2022; 40(3):111106.

PMID: 35858577 PMC: 9345261. DOI: 10.1016/j.celrep.2022.111106.

Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2.

Cheung C, Huang T, Chow N, Zhang S, Zhao Y, Chau M J Cell Sci. 2022; 135(13).

PMID: 35635291 PMC: 9377714. DOI: 10.1242/jcs.259280.

GEF-independent Ran activation shifts a fraction of the protein to the cytoplasm and promotes cell proliferation.

Zhou J, Tan Y, Zhang Y, Tong A, Shen X, Sun X Mol Biomed. 2022; 1(1):18.

PMID: 35006421 PMC: 8607414. DOI: 10.1186/s43556-020-00011-2.

Measuring and Interpreting Nuclear Transport in Neurodegenerative Disease-The Example of C9orf72 ALS.

Semmelink M, Steen A, Veenhoff L Int J Mol Sci. 2021; 22(17).

PMID: 34502125 PMC: 8431710. DOI: 10.3390/ijms22179217.

References

Kalderon D, Roberts B, Richardson W, Smith A . A short amino acid sequence able to specify nuclear location. Cell. 1984; 39(3 Pt 2):499-509. DOI: 10.1016/0092-8674(84)90457-4. View

Macara I . Transport into and out of the nucleus. Microbiol Mol Biol Rev. 2001; 65(4):570-94, table of contents. PMC: 99041. DOI: 10.1128/MMBR.65.4.570-594.2001. View

Fahrenkrog B, Koser J, Aebi U . The nuclear pore complex: a jack of all trades?. Trends Biochem Sci. 2004; 29(4):175-82. DOI: 10.1016/j.tibs.2004.02.006. View

Jakel S, Albig W, Kutay U, Bischoff F, Schwamborn K, Doenecke D . The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1. EMBO J. 1999; 18(9):2411-23. PMC: 1171324. DOI: 10.1093/emboj/18.9.2411. View

Colbeau A, Nachbaur J, Vignais P . Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim Biophys Acta. 1971; 249(2):462-92. DOI: 10.1016/0005-2736(71)90123-4. View

Melchior F, Paschal B, Evans J, Gerace L . Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol. 1993; 123(6 Pt 2):1649-59. PMC: 2290879. DOI: 10.1083/jcb.123.6.1649. View

Lyman S, Guan T, Bednenko J, Wodrich H, Gerace L . Influence of cargo size on Ran and energy requirements for nuclear protein import. J Cell Biol. 2002; 159(1):55-67. PMC: 2173498. DOI: 10.1083/jcb.200204163. View

Paraskeva E, Izaurralde E, Bischoff F, Huber J, Kutay U, Hartmann E . CRM1-mediated recycling of snurportin 1 to the cytoplasm. J Cell Biol. 1999; 145(2):255-64. PMC: 2133107. DOI: 10.1083/jcb.145.2.255. View

Dingwall C, Laskey R . Nuclear targeting sequences--a consensus?. Trends Biochem Sci. 1991; 16(12):478-81. DOI: 10.1016/0968-0004(91)90184-w. View

10.

Weis K, Mattaj I, Lamond A . Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science. 1995; 268(5213):1049-53. DOI: 10.1126/science.7754385. View

11.

Kehlenbach R, Gerace L . Analysis of nuclear protein import and export in vitro using fluorescent cargoes. Methods Mol Biol. 2002; 189:231-45. DOI: 10.1385/1-59259-281-3:231. View

12.

Paine P, Moore L, Horowitz S . Nuclear envelope permeability. Nature. 1975; 254(5496):109-14. DOI: 10.1038/254109a0. View

13.

Melchior F . Nuclear protein import in a permeabilized cell assay. Methods Mol Biol. 1998; 88:265-73. DOI: 10.1385/0-89603-487-9:265. View

14.

Chan R, Forbes D . In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts. Methods Mol Biol. 2006; 322:289-300. DOI: 10.1007/978-1-59745-000-3_20. View

15.

Pemberton L, Paschal B . Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic. 2005; 6(3):187-98. DOI: 10.1111/j.1600-0854.2005.00270.x. View

16.

Pollard V, Michael W, Nakielny S, Siomi M, Wang F, Dreyfuss G . A novel receptor-mediated nuclear protein import pathway. Cell. 1996; 86(6):985-94. DOI: 10.1016/s0092-8674(00)80173-7. View

17.

Mosammaparast N, Pemberton L . Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol. 2004; 14(10):547-56. DOI: 10.1016/j.tcb.2004.09.004. View

18.

Adam S, Marr R, Gerace L . Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol. 1990; 111(3):807-16. PMC: 2116268. DOI: 10.1083/jcb.111.3.807. View

19.

Paschal B, Gerace L . Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62. J Cell Biol. 1995; 129(4):925-37. PMC: 2120498. DOI: 10.1083/jcb.129.4.925. View