title: Characterization of the nuclear import mechanism of the CCAAT-regulatory subunit Php4.
abstract: Php4 is a nucleo-cytoplasmic shuttling protein that accumulates in the nucleus during iron deficiency. When present in the nucleus, Php4 associates with the CCAAT-binding protein complex and represses genes encoding iron-using proteins. Here, we show that nuclear import of Php4 is independent of the other subunits of the CCAAT-binding complex. Php4 nuclear import relies on two functionally independent nuclear localization sequences (NLSs) that are located between amino acid residues 171 to 174 (KRIR) and 234 to 240 (KSVKRVR). Specific substitutions of basic amino acid residues to alanines within these sequences are sufficient to abrogate nuclear targeting of Php4. The two NLSs are biologically redundant and are sufficient to target a heterologous reporter protein to the nucleus. Under low-iron conditions, a functional GFP-Php4 protein is only partly targeted to the nucleus in imp1Δ and sal3Δ mutant cells. We further found that cells expressing a temperature-sensitive mutation in cut15 exhibit increased cytosolic accumulation of Php4 at the nonpermissive temperature. Further analysis by pull-down experiments revealed that Php4 is a cargo of the karyopherins Imp1, Cut15 and Sal3. Collectively, these results indicate that Php4 can be bound by distinct karyopherins, connecting it into more than one nuclear import pathway.
authors.affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Md Gulam Musawwir
authors.initials: MG
authors.lastname: Khan
authors.affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Jacques
authors.affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Jude
authors.initials: J
authors.lastname: Beaudoin
authors.affiliation: Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Simon
authors.initials: S
authors.lastname: Labbé
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D028901:alpha Karyopherins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics*
chemical_list: D023081:CCAAT-Binding Factor; C510481:Cut15 protein, S pombe; C510480:Imp1 protein, S pombe; D028884:Karyopherins; C519990:Php4 protein, S pombe; C000603654:Sal3 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins