Journal of Cell Science, Vol 108, Issue 5 1849-1861, Copyright © 1995 by Company of Biologists

JOURNAL ARTICLES

The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells

I Cserpan and A Udvardy
Biological Research Center, Hungarian Academy of Sciences, Szeged.

Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction. Phycoerythrine-NLS transport substrate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methyltransferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenously added cytosolic protein. All the characteristic properties of in vivo nuclear transport are faithfully reproduced with these transport substrates: (i) the transport requires a functional NLS in the transported protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear localization peptide efficiently competes for transport, while the transport-incompetent mutant peptide does not; and (iv) wheat germ agglutinin inhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or inefficient extraction of the cytosolic proteins during the permeabilization of the cells. The nuclear transport was proportional to the number of conjugated NLSs. Nuclear transport of phycoerythrine carrying 7-8 conjugated NLSs/subunit required the addition of exogenous cytosolic proteins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport. Nuclear transport with different combinations of transport substrates further supported the assumption that distinct transport mechanisms operate for different substrates. From a mixture of PE-NLS7-8 and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the absence of exogenous cytosol, while Bsp methyltransferase-NLS was efficiently transported. Exogenous cytosol promoted the nuclear transport of the highly conjugated substrate.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				K. J. Thompson, M. G. Fried, Z. Ye, P. Boyer, and J. R. Connor Regulation, mechanisms and proposed function of ferritin translocation to cell nuclei J. Cell Sci., May 15, 2002; 115(10): 2165 - 2177. [Abstract] [Full Text] [PDF]

				M. Lippai, L. Tirián, I. Boros, J. Mihály, M. Erdélyi, I. Belecz, E. Máthé, J. Pósfai, A. Nagy, A. Udvardy, et al. The Ketel Gene Encodes a Drosophila Homologue of Importin-{beta} Genetics, December 1, 2000; 156(4): 1889 - 1900. [Abstract] [Full Text]

				L. Tirián, J. Puro, M. Erdélyi, I. Boros, B. Papp, M. Lippai, and J. Szabad The KetelD Dominant-Negative Mutations Identify Maternal Function of the Drosophila Importin-{beta} Gene Required for Cleavage Nuclei Formation Genetics, December 1, 2000; 156(4): 1901 - 1912. [Abstract] [Full Text]

				I. C. Northwood, A. H. Y. Tong, B. Crawford, A. E. Drobnies, and R. B. Cornell Shuttling of CTP:Phosphocholine Cytidylyltransferase between the Nucleus and Endoplasmic Reticulum Accompanies the Wave of Phosphatidylcholine Synthesis during the G0 right-arrow G1 Transition J. Biol. Chem., September 10, 1999; 274(37): 26240 - 26248. [Abstract] [Full Text] [PDF]