Blm10 binds to pre-activated proteasome core particles with open gate conformation

Blm10 is bound to the yeast proteasome core particle, a crucial protease of eukaryotic cells [corrected]. Two gates, at both ends of the CP, control the access of protein substrates to the catalytic cavity of the CP. Normally, substrate access is auto-inhibited by a closed gate conformation unless regulatory complexes are bound to the CP and translocate protein substrates in an ATP-dependent manner. Here, we provide evidence that Blm10 recognizes pre-activated open gate CPs, which are assumed to exist in an equilibrium with inactive closed gate CP. Consequently, single-capped Blm10-CP shows peptide hydrolysis activity. Under conditions of disturbed CP assembly, as well as in open gate mutants, pre-activated CP or constitutively active CP, respectively, prevail. Then, Blm10 sequesters disordered and open gate CP by forming double-capped Blm10(2)-CP in which peptide hydrolysis activity is repressed. We conclude that Blm10 distinguishes between gate conformations and regulates the activation of CP.     

A role for transportin in the nuclear import of adenovirus core proteins and DNA

Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex. We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII. We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus. Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect. We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing. While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin alpha-importin beta, bacterially expressed GST-VII favours the transportin pathway. This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell. Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core.     

Phosphorylation of tyrosine 256 facilitates nuclear import of atypical protein kinase C

Herein, we employed a combined approach of molecular modeling and site-directed mutagenesis to address the role of tyrosine phosphorylation in transport of atypical protein kinase C (aPKC) into the nucleus. Computer modeling of the three-dimensional structure of the aPKC catalytic core, reveals that tyrosine 256 (Tyr256) is located at the lip of the activation loop and is conserved among members of the aPKC family, iota/lambda and zeta. Based on these findings, we examined whether tyrosine phosphorylation of aPKC on the activation lip may facilitate nuclear import. An antiserum was generated that selectively recognizes the phosphorylated Tyr256 residue in aPKC. By isolating nuclei of PC12 cells and immunoprecipitating aPKC with Ab-PY256, we observed that Tyr256 is rapidly phosphorylated upon NGF treatment prior to entry of aPKC into the nucleus. aPKC was observed to exclusively bind to importin-beta. The interaction between importin-beta and aPKC was enhanced upon tyrosine phosphorylation of aPKC and binding was abrogated when Tyr256 was mutated to phenylalanine. We propose that phosphorylation of aPKC at Tyr256 induces a conformation, whereby, the arginine-rich NLS is exposed, which then binds importin-beta leading to import of aPKC into the nucleus. Altogether, these findings document a novel role for the tyrosine phosphorylation in regulating import of atypical PKC into the nucleus.     

Cytosolic import factor- and Ran-independent nuclear transport of ribosomal protein L5

Ribosomal protein L5 is a shuttling protein that, in Xenopus oocytes, is involved in the nucleocytoplasmic transport of 5S rRNA. As demonstrated earlier, L5 contains three independent nuclear import signals (NLSs), which function in oocytes as well as in somatic cells. Upon physical separation, these NLSs differ in respect to their capacity to bind to nuclear import factors in vitro and to mediate the nuclear import of a heterologous RNP in vivo. As reported in this communication, analysis of the in vitro nuclear import activity of these three NLSs reveals that they also differ in respect to their requirements for cytosolic import factors and Ran. Nuclear import mediated by the N-terminal and the central NLS depends on cytosolic import factor(s) and Ran, whereas import via the C-terminal NLS occurs independently from these factors. Thus, the presence of multiple NLSs in ribosomal protein L5 appears to allow for efficient nuclear transport via utilisation of multiple, mechanistically different import pathways.     

Karyopherins and nuclear import

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.     

Computational identification of post‐translational modification‐based nuclear import regulations by characterizing nuclear localization signal‐import receptor interaction

The binding affinity between a nuclear localization signal (NLS) and its import receptor is closely related to corresponding nuclear import activity. PTM‐based modulation of the NLS binding affinity to the import receptor is one of the most understood mechanisms to regulate nuclear import of proteins. However, identification of such regulation mechanisms is challenging due to the difficulty of assessing the impact of PTM on corresponding nuclear import activities. In this study we proposed NIpredict, an effective algorithm to predict nuclear import activity given its NLS, in which molecular interaction energy components (MIECs) were used to characterize the NLS‐import receptor interaction, and the support vector regression machine (SVR) was used to learn the relationship between the characterized NLS‐import receptor interaction and the corresponding nuclear import activity. Our experiments showed that nuclear import activity change due to NLS change could be accurately predicted by the NIpredict algorithm. Based on NIpredict, we developed a systematic framework to identify potential PTM‐based nuclear import regulations for human and yeast nuclear proteins. Application of this approach has identified the potential nuclear import regulation mechanisms by phosphorylation of two nuclear proteins including SF1 and ORC6. Proteins 2014; 82:2783–2796. © 2014 Wiley Periodicals, Inc.     

A new role for nuclear transport factor 2 and Ran: nuclear import of CapG

The small GTPase Ran plays a central role in nucleocytoplasmic transport. Nuclear transport of Ran itself depends on nuclear transport factor 2 (NTF2). Here, we report that NTF2 and Ran control nuclear import of the filamentous actin capping protein CapG. In digitonin-permeabilized cells, neither GTPγS nor the GTP hydrolysis-deficient Ran mutant RanQ69L affect transit of CapG to the nucleus in the presence of cytosol. Obstruction of nucleoporins prevents nuclear transport of CapG, and we show that CapG binds to nucleoporin62. In addition, CapG interacts with NTF2, associates with Ran and is furthermore able to bind the NTF2–Ran complex. NTF2–Ran interaction is required for CapG nuclear import. This is corroborated by a NTF2 mutant with reduced affinity for Ran and a Ran mutant that does not bind NTF2, both of which prevent CapG import. Thus, a ubiquitously expressed protein shuttles to the nucleus through direct association with NTF2 and Ran. The role of NTF2 may therefore not be solely confined to sustaining the Ran gradient in cells.     

A microtubule-facilitated nuclear import pathway for cancer regulatory proteins

Nuclear protein import is dependent on specific targeting signals within cargo proteins recognized by importins (IMPs) that mediate translocation through the nuclear pore. Recent evidence, however, implicates a role for the microtubule (MT) network in facilitating nuclear import of the cancer regulatory proteins parathyroid hormone-related protein (PTHrP) and p53 tumor suppressor. Here we assess the extent to which MT and actin integrity may be generally required for nuclear protein import for the first time. We examine 10 nuclear-localizing proteins with diverse IMP-dependent nuclear import pathways, our results indicating that the cytoskeleton does not have a general mechanistic role in nuclear localization sequence-dependent nuclear protein import. Of the proteins examined, only the p110(Rb) tumor suppressor protein Rb, together with p53 and PTHrP, was found to require MT integrity for optimal nuclear import. Fluorescence recovery after photobleaching experiments indicated that the MT-dependent nuclear transport pathway increases both the rate and extent of Rb nuclear import but does not affect Rb nuclear export. Dynamitin overexpression experiments implicate the MT motor dynein in the import process. The results indicate that, additional to IMP/diffusion-dependent processes, certain cancer regulatory proteins utilize an MT-enhanced pathway for accelerated nuclear import that is presumably required for their nuclear functions.     

Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

To save or degrade: balancing proteasome homeostasis to maximize cell survival

Autophagic degradation of proteasomes (termed proteaphagy) is a conserved mechanism by which cells eliminate excess or damaged particles. This clearance is induced rapidly when organisms are starved for nitrogen and, because proteasomes are highly abundant, their breakdown likely makes an important contribution to the amino acid pools necessary for survival. By contrast, our recent studies found that proteasomes are not degraded in response to carbon starvation, even though bulk macroautophagy is similarly activated. Instead, carbon starvation induces sequestration of proteasomes into membrane-less cytoplasmic condensates previously termed proteasome storage granules (PSGs), which protect proteasomes from autophagic degradation. Preserving proteasomes in PSGs enhances the ability of yeast cells to recover from a variety of stresses, implying that rapid remobilization of stored proteasomes when conditions improve is advantageous to cell fitness. Consequently, the choice of whether to save or degrade proteasomes can profoundly impact cell survival.     

Sugar-dependent nuclear import of glycosylated proteins in living cells

The nuclear import of proteins larger than Mr 40,000 depends on the presence of a nuclear localization signal (NLS) corresponding either to a short peptide sequence or to defined sugars. The sugar-dependent nuclear import was previously evidenced by using glycosylated proteins (neoglycoproteins) introduced into the cytosol of cells either by electroporation or on digitonin-permeabilization and was shown to be distinct from the peptide NLS-mediated pathway. In this work, we used a microinjection approach to compare the two nuclear import pathways in intact living cells. The intracellular localization of fluorescent NLS-BSA or Glc-BSA injected into the cytosol was analyzed by confocal microscopy. Novel differences between the two mechanisms were evidenced. First, Glc-BSA migrated less efficiently into the nucleus than NLS-BSA because of a cytosolic retention. Second, the import of neoglycoproteins was not affected by microinjection of antinuclear import factor importin/karyopherin beta antibodies, whereas the NLS-dependent transport was completely abolished. Third, the nuclear import activity of Glc-BSA was found to be cell cycle-dependent in thymidine and hydroxyurea-treated HeLa cells, with greatest efficiency during G1/S transition and S phases, whereas NLS-BSA was imported with the same efficiency during any stage of the cell cycle but the G2 phase. Fourth, we show that after mitosis, nonglycosylated BSA was excluded from the nucleus contrary to Glc-BSA. In both cases, the nuclear import signals (NLS or alpha-glucoside) were grafted onto BSA; such tools led to a clear-cut conclusion, which will reach a full physiological significance when they are confirmed in the case of endogenous (glyco)proteins.     

Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.     

Influence of cargo size on Ran and energy requirements for nuclear protein import

Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.     

Active nuclear import of single-stranded oligonucleotides and their complexes with non-karyophilic macromolecules

The objective of this investigation was to characterize intranuclear accumulation of oligonucleotides and their adducts with non-karyophilic compounds in cultured animal cells and thus to present a model system for nucleic acid-mediated nuclear import. In digitonin-permeabilized cells, nuclear uptake of 3′-FITC-labeled, single-stranded 25-mer oligodeoxyribonucleotides was independent of added cytosolic protein, largely energy-dependent, inhibitable by wheat germ agglutinin but not by N-ethylmaleimide, and a function of their base composition. When coupled to FITC-labeled streptavidin or streptavidin-bovine serum albumin conjugates, the oligonucleotides delivered the proteins to the nuclear interior with rates roughly proportional to their karyophilicity as free molecules. Transport activity was also demonstrated for single-stranded oligoribonucleotides. The transport was energy-dependent, inhibited by GMP-PNP and wheat germ agglutinin, but unaffected by N-ethylmaleimide. Nuclear import of oligo(dG)₂₅/protein adducts needed 3 to 4 oligonucleotide signals per complex and the signal had to be at least 15 nucleotides long. Micro-injection experiments showed that the results obtained with digitonin-permeabilized cells are not artifacts of a quasi-intact cellular system. These data were confirmed by electron microscopy employing complexes of oligodeoxyribonucleotides with streptavidin-peroxidase-bovine serum albumin-1 nm gold. In permeabilized cells, the complexes docked to the cytoplasmic face of the nuclear pore complexes, were translocated through the central pore channel and accumulated in large quantities in the nuclear baskets before they were released into the nucleoplasm. These results demonstrate that nuclear uptake of oligonucleotides and their complexes is an active process mediated by nuclear pore complexes, which, at least regarding its cytoplasmic component, is different from the pathway requiring classical nuclear localization signals.     

Ceramide regulation of nuclear protein import

Nucleocytoplasmic trafficking is an essential and responsive cellular mechanism that directly affects cell growth and proliferation, and its potential to address metabolic challenge is incompletely defined. Ceramide is an antiproliferative sphingolipid found within vascular smooth muscle cells in atherosclerotic plaques, but its mechanism of action remains unclear. The hypothesis that ceramide inhibits cell growth through nuclear transport regulation was tested. In smooth muscle cells, exogenously supplemented ceramide inhibited classical nuclear protein import that involved the activation of cytosolic p38 mitogen-activated protein kinase (MAPK). After application of SB 202190, a specific and potent pharmacological antagonist of p38 MAPK, sphingolipid impingement on nuclear transport was corrected. Distribution pattern assessments of two essential nuclear transport proteins, importin-alpha and Cellular Apoptosis Susceptibility, revealed ceramide-mediated relocalization that was reversed upon the addition of SB 202190. Furthermore, cell counts, nuclear cyclin A, and proliferating cell nuclear antigen expression, markers of cellular proliferation, were diminished after ceramide treatment and effectively rescued by the addition of inhibitor. Together, these data demonstrate, for the first time, the sphingolipid regulation of nuclear import that defines and expands the adaptive capacity of the nucleocytoplasmic transport machinery.     

Characterization of chronic low-level proteasome inhibition on neural homeostasis

Increasing evidence suggests that proteasome inhibition plays a causal role in promoting the neurodegeneration and neuron death observed in multiple disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). The ability of severe and acute inhibition of proteasome function to induce neuron death and neuropathology similar to that observed in AD and PD is well documented. However, at present the effects of chronic low-level proteasome inhibition on neural homeostasis has not been elucidated. In order to determine the effects of chronic low-level proteasome inhibition on neural homeostasis, we conducted studies in individual colonies of neural SH-SY5Y cells that were isolated following continual exposure to low concentrations (100 nm) of the proteasome inhibitor MG115. Clonal cell lines appeared morphologically similar to control cultures but exhibited significantly different rates of both proliferation and differentiation. Elevated levels of protein oxidation and protein insolubility were observed in clonal cell lines, with all clonal cell lines being more resistant to neural death induced by serum withdrawal and oxidative stress. Interestingly, clonal cell lines demonstrated evidence for increased macroautophagy, suggesting that chronic low-level proteasome inhibition may cause an excessive activation of the lysosomal system. Taken together, these data indicate that chronic low-level proteasome inhibition has multiple effects on neural homeostasis, and suggests that studying the effects of chronic low-level proteasome inhibition may be useful in understanding the relationship between protein oxidation, protein insolubility, proteasome function, macroautophagy and neural viability in AD and PD.     

The dynamics and mechanisms of interleukin-1alpha and beta nuclear import

Pro-inflammatory members of the interleukin-1 (IL-1) family of cytokines (IL-1α and β) are important mediators of host defense responses to infection but can also exacerbate the damaging inflammation that contributes to major human diseases. IL-1α and β are produced by cells of the innate immune system, such as macrophages, and act largely after their secretion by binding to the type I IL-1 receptor on responsive cells. There is evidence that IL-1α is also a nuclear protein that can act intracellularly. In this study, we report that both IL-1α and IL-1β produced by microglia (central nervous system macrophages) in response to an inflammatory challenge are distributed between the cytosol and the nucleus. Using IL-1-β-galactosidase and IL-1-green fluorescent protein chimeras (analyzed by fluorescence recovery after photobleaching), we demonstrate that nuclear import of IL-1α is exclusively active, requiring a nuclear localization sequence and Ran, while IL-1β nuclear import is entirely passive. These data provide valuable insights into the dynamic regulation of intracellular cytokine trafficking.     

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.