GTP gamma S restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells.
Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finch et al., J Biol Chem 268, 5823-5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTP gamma S, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTP gamma S still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.
Finch
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Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA.
Chang
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DC
Chan
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D002466:Cell Nucleolus / Q000187:drug effects* / Q000378:metabolism*; D018274:Electroporation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D009173:Mycophenolic Acid / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D000090243:Nucleophosmin; D012335:RNA, Ribosomal / Q000096:biosynthesis
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D012335:RNA, Ribosomal; D000090243:Nucleophosmin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D009173:Mycophenolic Acid