Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin β cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin β during import. Our results indicate that the importin β transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin β moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin ␣/␤.Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257 GKISKH-WTGI 266 . This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin ␣. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin ␣, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin ␣.Nuclear transport is an active signal-mediated process that requires, in most cases, soluble transport factors and specific import signals. Two families of transport receptors have been identified in the importin ␤ superfamily, which is involved in both nuclear import and export, and the TAP superfamily that mediates nuclear export. Transport receptors recognize specific nuclear localization signals (NLSs) 1 and nuclear export signals exposed on the molecular surface of cargoes. In the classical nuclear import pathway proteins bearing a classical SV40-like NLS (PKKKRKV) are recognized by the importin ␣/␤ heterodimer (also known as karyopherin ␣/␤) (1-3). Importin ␣ (4) acts as an adaptor that recognizes NLS sequences after association with the receptor importin ␤ (5). The importin ␣/␤-NLS cargo complex is then translocated through the nuclear pore complex in a process that requires multiple rounds of interaction of the receptor importin ␤ with nucleoporins, likely via their exposed hydrophobic FG-rich motifs (6, 7

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, endofacial membrane protein originally identified based on its capacity to promote accelerated transbilayer phospholipid movement in response to Ca 2؉ . Recent evidence suggests that this protein also participates in cell response to various growth factors and cytokines, influencing myeloid differentiation, tumor growth, and the antiviral activity of interferon. Whereas plasma membrane PLSCR1 was shown to be required for normal recruitment and activation of Src kinase by stimulated cell surface growth factor receptors, PLSCR1 was also found to traffic into the nucleus and to tightly bind to genomic DNA, suggesting a possible additional nuclear function. We now report evidence that PLSCR1 directly binds to the 5-promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene (IP3R1) to enhance expression of the receptor. Probing a CpG island genomic library with PLSCR1 as bait identified four clones with avidity for PLSCR1, including a 191-bp fragment of the IP3R1 promoter. Using electrophoretic mobility shift and transcription reporter assays, the PLSCR1-binding site in IP3R1 was mapped to residues ؊101 GTAACCATGTGGA ؊89 , and the segment spanning Met 86 -Glu 118 in PLSCR1 was identified to mediate its transcriptional activity. The significance of this interaction between PLSCR1 and IP3R1 in situ was confirmed by comparing levels of IP3R1 mRNA and protein in matched cells that either expressed or were deficient in PLSCR1. These data suggest that in addition to its role at the plasma membrane, effects of PLSCR1 on cell proliferative and maturational responses may also relate to alterations in expression of cellular IP3 receptors. The phospholipid scramblase (PLSCR)2 gene family consists of an apparent tetrad of genes with identifiable orthologues conserved from Caenorhabditis elegans to man (1). The first identified member of this family (PLSCR1) was isolated based upon its capacity to promote Ca 2ϩ -dependent accelerated transbilayer membrane phospholipid (PL) movement, mimicking the remodeling of plasma membrane PL that is observed under conditions of injury and apoptosis (2, 3). PLSCR1 is a multiply palmitoylated, Ca 2ϩ -binding, Pro-and Cys-rich, endofacial plasma membrane protein that was shown to distribute into lipid raft domains and to be a substrate of the Abl and Src tyrosine kinases (4 -6). The exact biologic function of this protein remains controversial; although PLSCR1 mediates Ca 2ϩ -dependent transbilayer movement of PL in proteoliposomes (2, 3) and was reported to increase cell surface expression of phosphatidylserine through remodeling of plasma membrane PL in mammalian cells exposed to Ca 2ϩ ionophore and other inducers of injury or apoptosis (7-9), it has also been observed that induced elevation of PLSCR1 expression can occur without a detectable increase in PL movement between membrane leaflets, and gene deletion of PLSCR1 did not impair cell capacity to undergo this remodeling of cell surface PL (10 -12). Whereas its ro...

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.

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