Nuclear pore biogenesis into an intact nuclear envelope

Nuclear pore complexes (NPCs) serve as transport channels across the nuclear membrane, a double lipid bilayer that physically separates the nucleoplasm and cytoplasm of eukaryotic cells. New evidence suggests that the multiprotein nuclear pores also play a role in chromatin organization and gene expression. Given the importance of NPC function, it is not surprising that a growing list of human diseases and developmental defects have been linked to its malfunction. In order to fully understand the functional repertoire of NPCs and their essential role for nuclear organization, it is critical to determine the sequence of events that lead to the formation of nuclear pores. This is particularly relevant since NPC number, and possibly composition, are tightly linked to metabolic activity. Most of our knowledge is derived from NPC formation that occurs in dividing cells at the end of mitosis when the nuclear envelope (NE) and NPCs reform from disassembled precursors. However, NPC assembly also takes place during interphase into an intact NE. Importantly, this process is not restricted to dividing cells but also occurs during cell differentiation. Here, we will review aspects unique to this process, namely the regulation of nuclear expansion and the mechanisms of fusion between the outer and inner nuclear membranes. We will then discuss conserved and diverging mechanisms between post-mitotic and interphase assembly of the proteinaceous structure in light of recently published data.     

Nuclear import of spliceosomal snRNPs

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are the building units of the spliceosome. These RNA and protein complexes assemble in the cytoplasm. After proper assembly and RNA maturation, mature U snRNPs are imported into the cell nucleus, where they take part in the splicing process. In this paper we review the current knowledge of how U snRNPs enter the nucleus.     

Blm10 facilitates nuclear import of proteasome core particles

Short‐lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT‐like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP‐CP assemblies are primarily nuclear and promote cell division. During quiescence, RP‐CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10‐bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG‐rich nucleoporins and is dissociated from the CP by Ran‐GTP. Thus, Blm10 represents the first CP‐dedicated nuclear import receptor in yeast.     

PACemakers of proteasome core particle assembly

The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven alpha subunits. Subsequently, beta subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the beta7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of beta7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.     

Functional analysis of the proteasome regulatory particle

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from rpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.     

Nuclear import: A tale of two sites

The recently determined crystal structure of a nuclear localization sequence receptor has revealed an exquisitely specific interaction between ligand and receptor, and explains how simple and complex nuclear localization signals can both be recognized specifically by the same molecule.     

A gated channel into the proteasome core particle

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme.     

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.     

Nuclear transport: a guide to import receptors

After synthesis in the cytoplasm, nuclear proteins traverse the nuclear envelope as a result of the specific recognition of nuclear localization signals by import. Various approaches have now uncovered a range of proteins with at least some of the characteristics expected of import receptors. This article focuses on early steps in the nuclear import of proteins and surveys the recently identified candidate import receptors.     

Nuclear import of U snRNPs requires importin beta.

Macromolecules that are imported into the nucleus can be divided into classes according to their nuclear import signals. The best characterized class consists of proteins which carry a basic nuclear localization signal (NLS), whose transport requires the importin alpha/beta heterodimer. U snRNP import depends on both the trimethylguanosine cap of the snRNA and a signal formed when the Sm core proteins bind the RNA. Here, factor requirements for U snRNP nuclear import are studied using an in vitro system. Depletion of importin alpha, the importin subunit that binds the NLS, is found to stimulate rather than inhibit U snRNP import. This stimulation is shown to be due to a common requirement for importin beta in both U snRNP and NLS protein import. Saturation of importin beta-mediated transport with the importin beta-binding domain of importin alpha blocks U snRNP import both in vitro and in vivo. Immunodepletion of importin beta inhibits both NLS-mediated and U snRNP import. While the former requires re-addition of both importin alpha and importin beta, re-addition of importin beta alone to immunodepleted extracts was sufficient to restore efficient U snRNP import. Thus importin beta is required for U snRNP import, and it functions in this process without the NLS-specific importin alpha.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence

We have shown previously that protein kinase Cdelta (PKCdelta) is required for mitochondrial-dependent apoptosis. Here we show that PKCdelta is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCdelta. Mutation of the caspase cleavage site of PKCdelta inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCdelta catalytic fragment (CFdelta) in transfected cells results in nuclear localization and apoptosis. We show that the PKCdelta NLS is required for nuclear import of both full-length PKCdelta and CFdelta, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFdelta prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFdelta) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFdelta to protect against etoposide-induced apoptosis. These results indicate that PKCdelta regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.     

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.     

The base of the proteasome regulatory particle exhibits chaperone-like activity

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.