Bipartite Nuclear Localization Signal Research Articles

The Ran-GTP Gradient Spatially Regulates XCTK2 in the Spindle

Ran is a small GTP binding protein that was originally identified as a regulator of nucleocytoplasmic transport [1] and subsequently found to be important for spindle formation [2-5]. In mitosis, a gradient of Ran-GTP emanates from chromatin and diminishes toward spindle poles [6]. Ran-GTP promotes spindle self-organization through the release of importin-bound spindle assembly factors (SAFs), which stimulate microtubule (MT) nucleation and organization and regulate MT dynamics [7-9]. Although many SAFs are non-motile MT-associated proteins, such as NuMA, TPX2, and HURP [7, 10-12], Ran also controls motor proteins, including Kid and HSET/XCTK2 [13, 14]. The Kinesin-14 XCKT2 is important for spindle assembly and pole organization [15-20], and Ran-GTP is proposed to promote XCKT2 MT crosslinking activity by releasing importin α/β from a bipartite nuclear localization signal (NLS) located in the tail domain [14]. Here, we show that the Ran-GTP gradient spatially regulates XCTK2 within the spindle. A flattened Ran-GTP gradient blocked the ability of excess XCTK2 to stimulate bipolar spindle assembly and resulted in XCTK2-mediated bundling of free MTs. These effects required the XCTK2 tail, which promoted the motility of XCTK2 within the spindle independent of the Ran-GTP gradient. In addition, the turnover kinetics of XCTK2 were spatially controlled: they were faster near the poles relative to the chromatin, but not with a mutant XCTK2 that cannot bind to importin α/β. Our results support a model in which the Ran-GTP gradient spatially coordinates motor localization with motility to ensure efficient spindle formation.

Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear-cytoplasmic trafficking of cognate paramyxovirus M proteins that show a consistent nuclear trafficking phenotype.

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant's schizothoracin (Schizothorax prenanti).

Through the RT-PCR and rapid amplification of cDNA ends, two complementary deoxyribonucleic acid (cDNA) clones encoding heat-shock cognate 70 (HSC70, designated Sp-HSC70) and inducible heat-shock protein 70 (HSP70, designated Sp-HSP70) were isolated from the liver of Prenant's schizothoracin (Schizothorax prenanti). The cDNAs were 2344- and 2292-bp in length and contained 1950- and 1932-bp open reading frames, encoded proteins of 649 and 643 amino acids, respectively. Amino acid sequence analysis indicated that both Sp-HSC70 and Sp-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (an ATP-binding site motif, a bipartite nuclear targeting signal), and a cytoplasmic characteristic motif EEVD. Homology analysis revealed that Sp-HSC70 and Sp-HSP70 shared 77.5% identity and Sp-HSC70 shared more than 81.1% identity with the known HSC70s of other vertebrates, while Sp-HSP70 shared more than 77.5 % identity with the known HSP70s of other vertebrates. Fluorescent real-time quantitative RT-PCR showed that Sp-HSC70 and Sp-HSP70 mRNAs were found in all tested tissues, including blood, brain, heart, liver, spleen, head kidney, white muscle, skin, gonad, hypophysis, red muscle, and gill. The Sp-HSC70 and Sp-HSP70 mRNA expression level in blood and head kidney displayed a significant increase in vibrio-challenged group with the bacterium Aeromonas hydrophila at 24 h post-infection compared to a control group. Temporally, there was a clear time-dependent expression pattern of Sp-HSC70 or Sp-HSP70 gene after bacterial challenge, and the expression of Sp-HSC70 and Sp-HSP70 mRNAs reached a maximum level at 12 and 6 h post-challenge, respectively. Both returned to control level after 7 × 24 h. The results suggest that Sp-HSC70 and Sp-HSP70 genes may play important roles in mediating the immune responses of A. hydrophila-related diseases in the Prenant's schizothoracin.

A recently evolved isoform of the transcription factor BES1 promotes brassinosteroid signaling and development in Arabidopsis thaliana.

Brassinosteroids (BRs) are essential steroid hormones that regulate plant growth and development. The transcription factor BRI1-EMS-SUPPRESSOR1 (BES1) regulates the expression of thousands of target genes in response to BRs. Here, we report an Arabidopsis thaliana-specific long isoform of BES1, BES1-L, which has stronger activity in promoting BR signaling than the canonical and widely used short BES1-S. The BES1-L isoform contains an additional N-terminal bipartite nuclear localization signal, which strongly promotes its nuclear localization. BES1-L also promotes the nuclear localization of BES1-S and BRASSINAZOLE-RESISTANT1 via dimerization. The transcription of BES1-L and BES1-S is differentially regulated by BRs due to the presence of G-box element in the BES1-S promoter. Moreover, BES1-L uniquely exists in the majority of A. thaliana ecotypes, but not in other species, even its Brassicaceae relatives, including Arabidopsis lyrata. The phenotypes of the BES1-L overexpression lines and plants with truncated BES1-L indicate that BES1-L is a more important isoform of BES1 in Arabidopsis and may have contributed to the evolution and expansion of A. thaliana.

Akt-mediated phosphorylation increases the binding affinity of hTERT for importin α to promote nuclear translocation.

Telomeres are essential for chromosome integrity and protection, and their maintenance requires the ribonucleoprotein enzyme telomerase. Previously, we have shown that human telomerase reverse transcriptase (hTERT) contains a bipartite nuclear localization signal (NLS; residues 222-240) that is responsible for nuclear import, and that Akt-mediated phosphorylation of residue S227 is important for efficient nuclear import of hTERT. Here, we show that hTERT binds to importin-α proteins through the bipartite NLS and that this heterodimer then forms a complex with importin-β proteins to interact with the nuclear pore complex. Depletion of individual importin-α proteins results in a failure of hTERT nuclear import, and the resulting cytoplasmic hTERT is degraded by ubiquitin-dependent proteolysis. Crystallographic analysis reveals that the bipartite NLS interacts with both the major and minor sites of importin-α proteins. We also show that Akt-mediated phosphorylation of S227 increases the binding affinity for importin-α proteins and promotes nuclear import of hTERT, thereby resulting in increased telomerase activity. These data provide details of a binding mechanism that enables hTERT to interact with the nuclear import receptors and of the control of the dynamic nuclear transport of hTERT through phosphorylation.

Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4).

Inactivating germline mutations of the CDKN1B gene encoding the nuclear cyclin-dependent kinase inhibitor P27kip1 protein have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to characterize in vitro the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by primary hyperparathyroidism due to multiglandular parathyroid involvement and gastro–entero–pancreatic tumors. We carried out subcellular localization experiments by transfection with plasmid vectors expressing the WT or mutant CDKN1B cDNA into the eukaryotic human cervix adenocarcinoma (HeLa) and GH3 cell lines. Results from western blotting studies indicated that fusion proteins were expressed at equal levels. The mutated protein was shorter compared with the WT protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells, WT P27 localized in the nucleus, whereas the P27_S125X protein was retained in the cytoplasm, predicting the loss of tumor-suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, because both WT and mutant alleles were determined to be present by sequencing the somatic DNA. Immunohistochemistry revealed a complete loss of nuclear expression of P27 in a parathyroid adenoma, which had been removed by the second surgery in the patient. In conclusion, our results confirm the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4.

The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum.

During development, Schistosoma japonicum undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Proteins containing zinc finger motifs usually play an important role in DNA recognition, RNA packaging, and transcriptional activation. In our current study, we cloned the open reading frame (ORF) of SjZFP1 of S. japonicum, which encodes a zinc finger protein. We analyzed the complementary DNA (cDNA) sequence of SjZFP1 and examined the expression of SjZFP1 messenger RNA (mRNA) at various developmental stages. We also tested the effects of RNA interference (RNAi) silencing on worm burden, spawning, and egg hatching. The ORF in the SjZFP1 cDNA was 1017 bp in length and was predicted to encode a 338-aa protein with a molecular mass of approximately 38.5 kDa and theoretical isoelectric point (pI) of 7.08. Several conserved regions, including a B-box-type zinc-binding domain, two bipartite nuclear localization signal domains, a paired amphipathic helix repeat, and overlapping RING and PHD finger domains, were identified in the predicted amino acid sequence of SjZFP1. Using real-time PCR, we showed that the SjZFP1 mRNA was expressed across all of the developmental stages of the parasite and that the level of transcription was highest in the cercariae, eggs, schistosomula, and mature adult worms. The level of SjZFP1 mRNA expression in cultured schistosomula treated with one of two SjZFP1-specific small interfering RNAs (siRNAs; AY770 and AY546) was reduced by over 80 %, compared with that in the controls. In RNAi experiments in BALB/c mice, the level of SjZFP1 mRNA increased significantly when the mice were treated with the same SjZFP1-specific siRNAs during the early stages of infection. By contrast, the level of SjZFP1 mRNA decreased significantly when the mice were treated with the SjZFP1-specific siRNAs during the middle to late stages of infection. In four independent experiments, fewer worms were recovered from mice treated with the SjZFP1-specific siRNAs, compared with the number of worms recovered from the control mice. Both the average number and hatching rates of liver eggs recovered from mice treated with the SjZFP1-specific siRNAs during the middle to late stages of infection were significantly lower than those of the liver eggs recovered from the control mice. Our results suggest that the SjZFP1 gene might be important for parasite development, spawning in the vertebrate host, and egg hatching.

A putative pH-dependent nuclear localization signal in the juxtamembrane region of c-Met.

The C-terminal fragment of the c-Met receptor tyrosine kinase is present in the nuclei of some cells irrespective of ligand stimulation, but the responsible nuclear localization signal (NLS) has not been previously reported. Here, we report that two histidine residues separated by a 10-amino-acid spacer (H1068–H1079) located in the juxtamembrane region of c-Met function as a putative novel NLS. Deletion of these sequences significantly abolished the nuclear translocation of c-Met, as did substitution of the histidines with alanines. This substitution also decreased the association of c-Met fragment with importin β. The putative NLS of c-Met is unique in that it relies on histidines, whose positive charge changes depending on pH, rather than the lysines or arginines, commonly found in classical bipartite NLSs, suggesting the possible ‘pH-dependency' of this NLS. Indeed, decreasing the cytosolic pH either with nigericin, an Na+/H+ exchanger or pH 6.5 KRB buffer significantly increased the level of nuclear c-Met and the interaction of the c-Met fragment with importin β, indicating that low pH itself enhanced nuclear translocation. Consistent with this, nigericin treatment also increased the nuclear level of endogenous c-Met in HeLa cells. The putative aberrant bipartite NLS of c-Met seems to be the first example of what we call a ‘pH-dependent' NLS.

Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.

In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

Functional complementation in Drosophila to predict the pathogenicity of TARDBP variants: evidence for a loss-of-function mechanism

The human TAR DNA binding protein 43 (TDP-43), encoded by the gene TARDBP, plays a central role in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. TDP-43 inclusions are also found in up to approximately 60% of Alzheimer's disease (AD) brains. Although ALS-causing TARDBP mutations cluster in the C-terminal glycine-rich region of the protein, the pathogenic nature of the atypical missense variants p.A90V (located between the bipartite nuclear localization signal) and p.D169G (located in the first RNA-binding domain) is unclear. In addition, whether causal ALS mutations represent gain or loss-of-function alleles remains unknown. We recently reported that loss-of-function of the highly conserved TARDBP ortholog in Drosophila (called TBPH) leads to death of bursicon neurons resulting in adult maturation and wing expansion defects. Here, we compared wild-type TARDBP, 2 typical ALS-causing mutations (p.G287S and p.A315T) and 2 atypical variants (p.A90V and p.D169G), for their ability to complement neuronal TBPH loss-of-function. Although p.D169G rescued organismal pupal lethality and neuronal loss to a similar extent as wild-type TARDBP, p.A90V, p.G287S, and p.A315T were less efficient. Accordingly, p.A90V, p.G287S, and p.A315T but not p.D169G or wild-type protein promoted a shift of TDP-43 from the nucleus to the cytoplasm in approximately 12%–14% of bursicon neurons. Finally, we found that the carrier frequency of rare variant p.A90V was higher in French-Belgian AD cases (5/1714, 0.29%) than in controls of European descent (5/9436, 0.05%) (odds ratio = 5.5; 95% confidence interval, 1.6–19.0; p = 0.009). We propose that pathogenic TARDBP mutations have partial loss-of-function properties and that TARDBP p.A90V may increase AD risk by the same mechanism.

Beak and feather disease viruses circulating in Cape parrots (Poicepahlus robustus) in South Africa.

Captive and wild psittacines are vulnerable to the highly contagious psittacine beak and feather disease. The causative agent, beak and feather disease virus (BFDV), was recently detected in the largest remaining population of endangered Cape parrots (Poicepahlus robustus), which are endemic to South Africa. Full-length genomes were isolated and sequenced from 26 blood samples collected from wild and captive Cape parrots to determine possible origins of infection. All sequences had characteristic BFDV sequence motifs and were similar in length to those described in the literature. However, BFDV coat protein (CP) sequences from this study did not contain a previously identified bipartite nuclear localisation signal (NLS) within residues 39-56, which indicates that an alternate NLS is involved in shuttling the CP into the nucleus. Sequences from the wild population shared a high degree of similarity, irrespective of year or location, suggesting that the disease outbreak occurred close to the time when the samples were collected. Phylogenetic analysis of full-length genomes showed that the captive Cape parrot sequences cluster with those isolated from captive-bred budgerigars in KwaZulu-Natal Province, South Africa. Exposure to captive-bred Cape parrots from a breeding facility in KwaZulu-Natal is suggested as a possible source for the virus infection. Phylogenetic analysis of BFDV isolates from wild and captive Cape parrots indicated two separate infection events in different populations, which highlights the potential risk of introducing new strains of the virus into the wild population. The present study represents the first systematic investigation of BFDV virus diversity in the southern-most population of Cape parrots.

Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus.

OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine(23) and arginine(24)) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins.

Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of porcine parvovirus capsid proteins.

Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. Importance: Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.

Autographa californica multiple nucleopolyhedrovirus DNA polymerase C terminus is required for nuclear localization and viral DNA replication.

The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.

Calpain 5 Is Highly Expressed in the Central Nervous System (CNS), Carries Dual Nuclear Localization Signals, and Is Associated with Nuclear Promyelocytic Leukemia Protein Bodies

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined.

Identification of a functional nuclear localization signal within the human USP22 protein

Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function.

Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin.

The small phytoplasma virulence effector SAP11 contains distinct domains required for nuclear targeting and CIN‐TCP binding and destabilization

Phytoplasmas are insect-transmitted bacterial phytopathogens that secrete virulence effectors and induce changes in the architecture and defense response of their plant hosts. We previously demonstrated that the small (± 10 kDa) virulence effector SAP11 of Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) binds and destabilizes Arabidopsis CIN (CINCINNATA) TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 AND 2) transcription factors, resulting in dramatic changes in leaf morphogenesis and increased susceptibility to phytoplasma insect vectors. SAP11 contains a bipartite nuclear localization signal (NLS) that targets this effector to plant cell nuclei.To further understand how SAP11 functions, we assessed the involvement of SAP11 regions in TCP binding and destabilization using a series of mutants.SAP11 mutants lacking the entire N-terminal domain, including the NLS, interacted with TCPs but did not destabilize them. SAP11 mutants lacking the C-terminal domain were impaired in both binding and destabilization of TCPs. These SAP11 mutants did not alter leaf morphogenesis. A SAP11 mutant that did not accumulate in plant nuclei (SAP11ΔNLS-NES) was able to bind and destabilize TCP transcription factors, but instigated weaker changes in leaf morphogenesis than wild-type SAP11.Overall the results suggest that phytoplasma effector SAP11 has a modular organization in which at least three domains are required for efficient CIN-TCP destabilization in plants.

Residues R199H200 of prototype foamy virus transactivator Bel1 contribute to its binding with LTR and IP promoters but not its nuclear localization

Prototype foamy virus encodes a transactivator called Bel1 that enhances viral gene transcription and is essential for PFV replication. Nuclear localization of Bel1 has been reported to rely on two proximal basic motifs R199H200 and R221R222R223 that likely function together as a bipartite nuclear localization signal. In this study, we report that mutating R221R222R223, but not R199H200, relocates Bel1 from the nucleus to the cytoplasm, suggesting an essential role for R221R222R223 in the nuclear localization of Bel1. Although not affecting the nuclear localization of Bel1, mutating R199H200 disables Bel1 from transactivating PFV promoters. Results of EMSA reveal that the R199H200 residues are vital for the binding of Bel1 to viral promoter DNA. Moreover, mutating R199H200 in Bel1 impairs PFV replication to a much greater extent than mutating R221R222R223. Collectively, our findings suggest that R199H200 directly participate in Bel1 binding to viral promoter DNA and are indispensible for Bel1 transactivation activity.