Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein

The nucleolar protein PICT1 regulates tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2, leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine, or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked PICT1 degradation, whereas the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation fail to repress PICT1 degradation. In addition, the 20 S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, although the same mutant underwent in vitro degradation by the 20 S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1 degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar stress within the nucleolus.     

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

ABSTRACT: BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.     

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-Like Potato virus A in Nicotiana Species

The multifunctional nuclear inclusion protein a (NIa) of potyviruses (genus Potyvirus; Potyviridae) accumulates in the nucleus of virus-infected cells for unknown reasons. In this study, two regions in the viral genome-linked protein (VPg) domain of NIa in Potato virus A (PVA) were found to constitute nuclear and nucleolar localization signals (NLS) in plant cells (Nicotiana spp). Amino acid substitutions in both NLS I (residues 4 to 9) and NLS II (residues 41 to 50) prevented nuclear localization, whereas mutations in either single NLS did not. Mutations in either NLS, however, prevented nucleolar localization and prevented or diminished virus replication in protoplasts, accumulation in infected plant tissues, and/or systemic movement in plants. One NLS mutant was partially complemented by the wild-type VPg expressed in transgenic plants. Furthermore, NLS I controlled NIa accumulation in Cajal bodies. The VPg domain interacted with fibrillarin, a nucleolar protein, and depletion of fibrillarin reduced PVA accumulation. Overexpression of VPg in leaf tissues interfered with cosuppression of gene expression (i.e., RNA silencing), whereas NLS I and NLS II mutants, which exhibited reduced nuclear and nucleolar localization, showed no such activity. These results demonstrate that some of the most essential viral functions required for completion of the infection cycle are tightly linked to regulation of the NIa nuclear and nucleolar localization.     

Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.     

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.     

Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.

To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture.     

Specific sequences within arginine–glycine-rich domains affect mRNA-binding protein function

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine–glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation.     

Charge versus sequence for nuclear/nucleolar localization of plant ribosomal proteins

Ribosomal subunit assembly in the nucleolus is dependent on efficient targeting of ribosomal proteins (RPs) from the cytoplasm into the nucleus and nucleolus. Nuclear/nucleolar localization of a protein is generally mediated by one or more specific stretches of basic amino acids—nuclear/nucleolar localization signals (NLSs/NoLSs). Arabidopsis thaliana RPL23aA has eight putative NLSs/NoLSs (pNLSs/NoLSs). Here we mutated all eight NLS/NoLSs individually and in groups and showed, via transient expression in tobacco cells that nucleolar localization of RPL23aA was disrupted by mutation of various combinations of five or more pNLSs/NoLSs. Mutation of all eight pNLSs/NoLSs, a 50 % reduction in total basic charge of RPL23aA, resulted in a complete disruption of nucleolar localization, however, the protein can still localize to the nucleus. As no individual or specific combination of NoLSs was absolutely required for nucleolar localization, we suggest that nucleolar localization/retention of RPL23aA is dependent on the overall basic charge. In addition to the optimal basic charge conferred by these NoLSs, nucleolar localization/retention of RPL23aA also required a C-terminal putative 26S rRNA binding site. In contrast, in the RPs RPS8A and RPL15A, mutation of just two and three N-terminal pNLSs, respectively, disrupted both nuclear and nucleolar localization of these two RPs, indicating differential signal requirements for nuclear and nucleolar localization of the three Arabidopsis RPs RPL23aA, RPL15A and RPS8A.     

Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B–12 (S10B–12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts.     

Mmi1, the Yeast Homologue of Mammalian TCTP,Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.     

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.     

Mechanism of cullin3 e3 ubiquitin ligase dimerization

Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.     

Nucleolin interacts with US11 protein of herpes simplex virus 1 and is involved in its trafficking

Herpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection.     

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).