Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr.

BACKGROUND: Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies 1 involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins alpha and beta from Xenopus laevis egg extracts 2, although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus34. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin beta and importin alpha by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay. RESULTS: We found that Tpr binds strongly and specifically to importin alpha, importin beta, and a CRM1 containing trimeric export complex, and that the binding sites for importins alpha and beta are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin alpha/beta import pathway. CONCLUSION: Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin alpha/beta heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin beta during import of importin beta cargo complexes and/or importin beta recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.

Phospholipid scramblase 1 binds to the promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene to enhance its expression.

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.

Phospholipid scramblase 1 contains a nonclassical nuclear localization signal with unique binding site in importin alpha.

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin alpha/beta. 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)GKISKHWTGI(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 A crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin alpha. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin alpha, 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 alpha.

Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA.

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.