Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C delta1 (original) (raw)
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Journal of Cell Biology, 2002
he nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cyto-T plasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin ␣ /  -or transportin-dependent import.
The Journal of Cell Biology, 1992
We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol . NBPs are phosphorylated and phosphatase treatment abolished NLS binding . The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells . Proper import of T HE composition of the nucleus requires vectorial transport of macromolecules across the nuclear envelope. Thus far, import of proteins into the nucleus could be divided into several steps . The initial reaction includes specific recognition and binding of nuclear proteins at the nuclear envelope . This is followed by ATP-and temperaturedependent translocation through the nuclear pore complex .
Molecular and cellular biology, 1989
The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unla...
Genetic Analysis of Macromolecular Transport across the Nuclear Envelope
Experimental Cell Research, 1996
bind at the cytoplasmic surface of the NPC [11, 12]. Numerous factors that promote movement of macro-Following binding, vectorial movement through the molecules in and out of the nucleus have now been pore complex is catalyzed by a host of factors. These identified. These include both soluble cytoplasmic and factors include a GTPase, termed Ran, and its various nucleoplasmic proteins and proteins of the nuclear regulators, including a GAP and an exchange factor pore complex (NPC). Genetic analyses of the nuclear [13][16]. In addition, proteins of the NPC are intitransport process in the model organism, the budding mately involved in the import process [18][19][20][21]. The acyeast Saccharomyces cerevisiae, have revealed retual manner by which GTP promotes import is under markable conservation of all of these factors. In addiintense study. However, the facts that the GAP, termed tion, important clues as to how these factors promote Rna1p, is located primarily in the cytoplasm [22] and the unique bidirectional movement across the NPC the exchange factor, termed Rcc1, is nuclear [23] prohave emerged from studies of yeast. We summarize the vide important clues around which various models characterization and genetic interactions of the soluhave been constructed and tested.
Journal of Biological Chemistry, 2011
Importin ␣1 can bind classical nuclear localization signals (NLSs) in two NLS-binding sites, known as "major" and "minor." The major site is located between ARM repeats 2-4, whereas the minor site spans ARM 7-8. In this study, we have characterized the cellular localization of human phospholipid scramblase 4 (hPLSCR4), a member of the phospholipid scramblase protein family. We identified a minimal NLS in hPLSCR4 (273 GSIIRKWN 280) that contains only two basic amino acids. This NLS is both necessary for nuclear localization of hPLSCR4 in transfected HeLa cells and sufficient for nuclear import of a non-diffusible cargo in permeabilized cells. Mutation of only one of the two basic residues, Arg 277 , correlates with loss of nuclear localization, suggesting this amino acid plays a key role in nuclear transport. Crystallographic analysis of mammalian importin ␣1 in complex with the hPLSCR4-NLS reveals this minimal NLS binds specifically and exclusively to the minor binding site of importin ␣. These data provide the first structural and functional evidence of a novel NLS-binding mode in importin ␣1 that uses only the minor groove as the exclusive site for nuclear import of nonclassical cargos.