Sugar-dependent nuclear import of glycosylated proteins in living cells.
The nuclear import of proteins larger than Mr 40,000 depends 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 on 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 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 antinuclear import factor importin/karyopherin beta antibodies, whereas 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 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, nonglycosylated 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 are confirmed in the case of endogenous (glyco)proteins.
Rondanino
Christine
C
Glycobiologie, Vectorologie et Trafic Intracellulaire, Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, F-45071 Orléans Cedex 2, France. rondanin@cnrs-orleans.fr
Bousser
Marie-Therese
MT
Monsigny
Michel
M
Roche
Annie-Claude
AC
D021581:Active Transport, Cell Nucleus; D050260:Carbohydrate Metabolism; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D028884:Karyopherins / Q000378:metabolism; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*
D028884:Karyopherins; D011506:Proteins; D015739:Nocodazole; D006918:Hydroxyurea