Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import.
Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.
Cell and Molecular Biology Program, Michigan State University, East Lansing, MI 48824, USA.
Peter J
PJ
Davidson
Su-Yin
SY
Li
Andrew G
AG
Lohse
Rianna
R
Vandergaast
Elisa
E
Verde
Andrea
A
Pearson
Ronald J
RJ
Patterson
John L
JL
Wang
Eric J
EJ
Arnoys
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D037502:Galectin 3 / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
D037502:Galectin 3; D019913:Nuclear Localization Signals; D049452:Green Fluorescent Proteins