title: Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA.
abstract: Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, Ca(2+)-binding, endofacial plasma membrane protein originally identified by its capacity to accelerate transbilayer movement of membrane phospholipids. We recently reported that when palmitoylation of PLSCR1 does not occur, it is localized to the nucleus rather than the plasma membrane. Nuclear localization of PLSCR1 was also observed upon induction of its de novo synthesis by cytokines such as interferon alpha that activate the PLSCR1 gene. Despite its capacity to enter the nucleus, its sequence does not predict a nuclear localization signal. To gain insight into the mechanism and potential significance of nuclear PLSCR1, we investigated the conditions required for its import and retention in the nucleus. We show that nuclear localization of PLSCR1 is dependent on cytosolic factors and energy. Furthermore, we show that PLSCR1 is specifically transported into the nucleus by the importin alpha/beta import pathway, and binds directly and with high affinity to importin alpha. Analysis of deletion mutants suggested that the NLS of PLSCR1 is between residues 242 and 290 and, furthermore, that a peptide within this region encompassing residues (257)GKISKHWTGI(266) is sufficient for nuclear import when conjugated to BSA. In addition, in intact cells, mutation of positively charged amino acids within this putative NLS in the full-length protein completely blocked its entry into the nucleus, consistent with its role in targeting PLSCR1 to the nucleus. Release of PLSCR1 from the nucleus was only observed after treatment of cells with both detergent and an elevated NaCl concentration, or following DNase treatment of the nucleus, suggesting ionic interactions of PLSCR1 with a nuclear component bound to genomic DNA or directly with genomic DNA. Purified PLSCR1 was also found to bind directly to a genomic DNA-cellulose conjugate, and its elution from DNA also required an elevated NaCl concentration. These data support a mechanism of receptor-mediated nuclear import of PLSCR1 and suggest a potential nuclear function for this plasma membrane protein.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Iris
authors.initials: I
authors.lastname: Ben-Efraim
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Zhou
authors.forename: Therese
authors.initials: T
authors.lastname: Wiedmer
authors.forename: Larry
authors.initials: L
authors.lastname: Gerace
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Sims
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D017830:Octoxynol; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology; D049294:Phospholipid Transfer Proteins; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D028901:alpha Karyopherins / Q000378:metabolism / Q000502:physiology; D028961:beta Karyopherins / Q000502:physiology; D020931:ran GTP-Binding Protein / Q000502:physiology
chemical_list: D002021:Buffers; D002352:Carrier Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D049294:Phospholipid Transfer Proteins; D011956:Receptors, Cell Surface; D028901:alpha Karyopherins; D028961:beta Karyopherins; D017830:Octoxynol; D004247:DNA; D020931:ran GTP-Binding Protein