Search

Abstract 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 α and β 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 β and importin α 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 α, importin β, and a CRM1 containing trimeric export complex, and that the binding sites for importins α and β 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 α/β import pathway. Conclusion Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin α/β heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin β during import of importin β cargo complexes and/or importin β recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.

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 (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.

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 (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.

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 beta 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 beta during import. Our results indicate that the importin beta 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 beta 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.

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low alpha-helical structure. However, when the two peptides are mixed together, their alpha-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into alpha-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion., (Copyright 1999 Academic Press.)

The hydropathy plot of the inwardly rectifying ROMK1 K+ channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post-M2). Peptides that correspond to M0, post-M2, and a control peptide, pre-M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self-assemble and coassemble in the membrane-bound state. CD spectroscopy revealed that both M0 and post-M2 adopt highly alpha-helical structures in 1% SDS and 40% TFE/water, whereas pre-M0 is not alpha-helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD-labeled peptides demonstrated that both M0 and post-M2, but not pre-M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10(3)-10(5) M-1. A surface localization for both post-M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/ acceptor (rhodamine) peptides revealed that M0 and post-M2 can coassemble in their membrane-bound state, but cannot self-associate when membrane-bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K+ channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994 Science 264:844-847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039-1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane-bound polypeptides are also discussed.

IsK, also referred to as minK, is a membrane protein consisting of 130 amino acids and localized mainly in epithelial cells but also in human T lymphocytes. Depending on the cRNA concentration that was injected into Xenopus oocytes, IsK and its truncated forms can induce either a K+ current alone or both K+ and Cl- currents [Attali et al. (1993) Nature 365, 850-852]. To obtain information on the secondary structure and the topology of IsK in a membrane-bound state, the synthesis, fluorescent-labeling, and structural and functional characterization of five polypeptides of 20-63 amino acids within the rat IsK protein were examined. The alpha-helical content of the segments, assessed in methanol using circular dichroism, suggests that both the N-terminal and transmembrane segments of IsK adopt alpha-helical structures. Binding experiments and the blue shift of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled peptides suggest that while both the alpha-helical transmembrane segment and the N-terminal of IsK are located within the lipid bilayer, the linking segment between the two segments lies on the surface of the membrane. The fluorescence energy transfer, between donor and acceptor-labeled truncated IsK, suggests that it aggregates within phospholipid membranes. Although a protein whose sequence is similar to that of truncated IsK can induce K+ channel activity when expressed in Xenopus oocytes, the inability of a truncated IsK to form functional K+ channels in planar lipid membranes supports increasing evidence that the protein alone cannot form a K+ channel.

MinK (Isk) is a voltage-dependent K+ channel whose gene has been recently cloned and which consists of 130 amino acids [Takumi, T., Ohkubo, H., & Nakanishi, S. (1988) Science 242, 1042-1045]. The protein contains one putative transmembrane segment by hydropathy analysis. Whether this putative transmembrane segment is involved in the function of the protein was studied. A 32 amino acid peptide (residues 41-72) with the sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing the hypothesized transmembrane domain, designed TM-minK, was synthesized and fluorescently labeled. The alpha-helical content of TM-minK, assessed in methanol using circular dichroism (CD), was 57%. The fluorescent emission spectrum of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed a blue shift upon binding to small unilamellar vesicles (SUV), reflecting a relocation of the fluorescent probe to an environment of increased apolarity, i.e., within the lipid bilayer. The increase in NBD's fluorescence upon mixing NBD-labeled TM-minK with small unilamellar vesicles (SUV) was used to generate a binding isotherm, from which was derived a surface partition coefficient of 5.5 x 10(4) M-1. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled and rhodamine-labeled analogues suggest that TM-minK aggregates within membranes. In addition, single-channel experiments revealed that TM-minK can form single channels in planar lipid membranes only when a trans negative potential is applied. The findings herein experimentally support a role of the transmembrane segment of minK both in the assembly and as a constituent of the pore formed by the protein.