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PMID: 17056062 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy.

Journal of molecular biology ·Vol. 365 ·No. 1 ·2007-01-05 ·Pages 50-65

Paradise A, Levin MK, Korza G, Carson JH

Abstract

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild-type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin alpha, importin beta, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.

MeSH Terms
Active Transport, Cell Nucleus Cell Nucleus/metabolism Cytoplasm/metabolism Humans Models, Molecular Nuclear Envelope/metabolism Nucleocytoplasmic Transport Proteins/chemistry,metabolism Pregnancy Proteins/chemistry,metabolism Recombinant Proteins/chemistry,metabolism Spectrometry, Fluorescence/instrumentation,methods Tumor Cells, Cultured alpha Karyopherins/chemistry,metabolism beta Karyopherins/chemistry,metabolism ran GTP-Binding Protein/chemistry,metabolism
Chemicals
NUTF2 protein, human Nucleocytoplasmic Transport Proteins Pregnancy Proteins Recombinant Proteins alpha Karyopherins beta Karyopherins ran GTP-Binding Protein
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Paradise Allison
Department of Molecular Microbial and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Levin Mikhail K
Korza George
Carson John H
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Article Info
Journal
Journal of molecular biology
Abbr.
J Mol Biol
ISSN
0022-2836
DOI
10.1016/j.jmb.2006.09.089
Published
2007-01-05
Epub
2006-00-04
Pages
50-65
Language
English
Region
England
NLM ID
2985088R
PMCID
PMC1831836
Subset
IM
Grants
NCRR NIH HHS · RR22232 · United States
NINDS NIH HHS · NS15190 · United States
NINDS NIH HHS · R56 NS015190 · United States
NCRR NIH HHS · P41 RR013186 · United States
NCRR NIH HHS · S10 RR022624 · United States
NINDS NIH HHS · R01 NS015190 · United States
NCRR NIH HHS · U54 RR022232 · United States
NCRR NIH HHS · RR13186 · United States
External Links
PubMed source DOI: 10.1016/j.jmb.2006.09.089 PMC Full Text View journal details
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