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Glycobiology Advance Access published online on April 2, 2003
Glycobiology, doi:10.1093/glycob/cwg064
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Submitted on August 6, 2002
Revised on February 27, 2003
Accepted on March 4, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Sugar-dependent nuclear import of glycosylated proteins in living cells

Christine Rondanino 1*, Marie-Thérèse Bousser 1, Michel Monsigny 1, Annie-Claude Roche 1

1 Glycobiologie, Vectorologie et Trafic intracellulaire, Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, F-45071 Orléans Cedex 2, France

* To whom correspondence should be addressed. E-mail: rondanin{at}cnrs-orleans.fr.

Abstract

The nuclear import of proteins larger than Mr 40,000 depends either on the presence of a nuclear localization signal (NLS) corresponding either to a short peptide sequence or to defined sugars. The sugar-dependent nuclear import was previously evidenced by using glycosylated proteins (neoglycoproteins) introduced into the cytosol of cells either by electroporation or upon digitonin-permeabilization and was shown to be distinct from the peptide NLS-mediated pathway.

In this work, we used a microinjection approach to compare the two nuclear import pathways in intact living cells. The intracellular localization of fluorescent NLS-BSA or Glc-BSA (BSA substituted with {alpha}-glucoside moieties) injected into the cytosol was analyzed by confocal microscopy. Novel differences between the two mechanisms were evidenced. First, Glc-BSA migrated less efficiently into the nucleus than NLS-BSA because of a cytosolic retention. Second, the import of neoglycoproteins was not affected by microinjection of anti-nuclear import factor importin/karyopherin {beta} antibodies while the NLS-dependent transport was completely abolished. Third, the nuclear import activity of Glc-BSA was found to be cell cycle-dependent in thymidine and hydroxyurea-treated HeLa cells, with a greatest efficiency during G1/S transition and S phases whereas NLS-BSA was imported with the same efficiency during any stage of the cell cycle, but the G2 phase. Fourth, we show that, after mitosis, non-glycosylated BSA was excluded from the nucleus contrary to Glc-BSA. In both cases, the nuclear import signals (NLS or {alpha}-glucoside) were grafted onto BSA; such tools led to a clear cut conclusion, which will reach a full physiological significance when they will be confirmed in the case of endogenous (glyco)proteins.


karyophilic sugars, cell synchronization, microinjection, nuclear import
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