Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

作者：Kakuk Annamaria; Friedlander Elza; Vereb Gyoergy Jr; Li**oa Duarte; Bagossi Peter; Toth Gabor; Gergely Pal; Vereb Gyoergy

来源：Experimental Cell Research, 2008, 314(13): 2376-2388.

DOI：10.1016/j.yexcr.2008.05.006

• phosphatidylinositol 4-kinase
• PI4K230
• nuclear localization signal
• NLS
• nucleolar targeting
• NTS
• importins
• permeabilized HeLa cells
• import assay

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 alpha 1 and alpha 3 but not alpha 5. This transport was inhibited by wheat germ agglutinin and GTP gamma S. 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 alpha 1/beta or alpha 3/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.

• 出版日期2008-8-1
• 单位河北医科大学