Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230.
PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence (916)NFNHIHKRIRRVADKYLSG(934) comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin alpha/beta mechanism employing importins alpha1 and alpha3 but not alpha5. This transport was inhibited by wheat germ agglutinin and GTPgammaS. The sequence (1414)SKKTNRGSQLHKYYMKRRTL(1433), a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin alpha1/beta or alpha3/beta complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.
Kakuk
Annamária
A
Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, H-4032 Debrecen, Nagyerdei krt. 98, Hungary.
Friedländer
Elza
E
Vereb
György
G
Lisboa
Duarte
D
Bagossi
Péter
P
Tóth
Gábor
G
Gergely
Pál
P
Vereb
György
G
D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D028884:Karyopherins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D021381:Protein Transport; D018411:Spodoptera
D007527:Isoenzymes; D028884:Karyopherins; D019913:Nuclear Localization Signals; D019870:1-Phosphatidylinositol 4-Kinase