Nuclear Import Mechanism Research Articles

Shuttling from the extracellular matrix to the nucleus

Several enzymes secreted in the extracellular space, such as matrix metalloproteinases and lysyl oxidase, are internalized and translocated to the nucleus, where they may act as proteases and transcription factors to regulate gene expression and enhance apoptosis. Membrane proteoglycan syndecans, glycosaminoglycans and an anti-angiogenic matricryptin of collagen XVIII have also been identified in the nucleus. The nuclear entry of most extracellular proteins is likely mediated by nuclear localizing sequences. The molecular mechanisms of nuclear import, the physiopathological contexts, which induce it, and the biological roles played in vivo by extracellular proteins and proteoglycans are still underexplored.

Issue Cover (January 2016)

Front cover: The photomicrographs show both the co-localization of the glioblastoma tumor suppressor neurofibromin with mitotic microtubules and normally congressed chromosomes at metaphase in astrocytes. Neurofibromin depletion leads to aberrant chromosome congression, a possible mechanism for the generation of CIN/aneuploidy in neurofibromatosis patients who have a 5-fold increase incidence of glioblastoma. Read the full article ‘Nuclear import mechanism of neurofibromin for localization on the spindle and function in chromosome congression’ by X. Koliou, C. Fedonidis, T. Kalpachidou and D. Mangoura (J. Neurochem. 2016, vol. 136 (1), pp. 78–91) on doi: 10.1111/jnc.13401

Nuclear import mechanism of neurofibromin for localization on the spindle and function in chromosome congression.

Neurofibromatosis type-1 (NF-1) is caused by mutations in the tumor suppressor gene NF1; its protein product neurofibromin is a RasGTPase-activating protein, a property that has yet to explain aneuploidy, most often observed in astrocytes in NF-1. Here, we provide a mechanistic model for the regulated nuclear import of neurofibromin during the cell cycle and for a role in chromosome congression. Specifically, we demonstrate that neurofibromin, phosphorylated on Ser2808, a residue adjacent to a nuclear localization signal in the C-terminal domain (CTD), by Protein Kinase C-epsilon (PKC-ε), accumulates in a Ran-dependent manner and through binding to lamin in the nucleus at G2 in glioblastoma cells. Furthermore, we identify CTD as a tubulin-binding domain and show that a phosphomimetic substitution of its Ser2808 results in a predominantly nuclear localization. Confocal analysis shows that endogenous neurofibromin localizes on the centrosomes at interphase, as well as on the mitotic spindle, through direct associations with tubulins, in glioblastoma cells and primary astrocytes. More importantly, analysis of mitotic phenotypes after siRNA-mediated depletion shows that acute loss of this tumor suppressor protein leads to aberrant chromosome congression at the metaphase plate. Therefore, neurofibromin protein abundance and nuclear import are mechanistically linked to an error-free chromosome congression. Concerned with neurofibromin's, a tumor suppressor, mechanism of action, we demonstrate in astrocytic cells that its synthesis, phosphorylation by Protein Kinase C-ε on Ser2808 (a residue adjacent to a nuclear localization sequence), and nuclear import are cell cycle-dependent, being maximal at G2. During mitosis, neurofibromin is an integral part of the spindle, while its depletion leads to aberrant chromosome congression, possibly explaining the development of chromosomal instability in Neurofibromatosis type-1. Read the Editorial Highlight for this article on page 11. Cover Image for this issue: doi: 10.1111/jnc.13300.

Karyopherin α 3 and Karyopherin α 4 Proteins Mediate the Nuclear Import of Methyl-CpG Binding Protein 2

Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients.

Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.

Drosophila TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus

Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In Drosophila, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER.

Characterization of the nuclear import mechanism of the CCAAT-regulatory subunit Php4.

Php4 is a nucleo-cytoplasmic shuttling protein that accumulates in the nucleus during iron deficiency. When present in the nucleus, Php4 associates with the CCAAT-binding protein complex and represses genes encoding iron-using proteins. Here, we show that nuclear import of Php4 is independent of the other subunits of the CCAAT-binding complex. Php4 nuclear import relies on two functionally independent nuclear localization sequences (NLSs) that are located between amino acid residues 171 to 174 (KRIR) and 234 to 240 (KSVKRVR). Specific substitutions of basic amino acid residues to alanines within these sequences are sufficient to abrogate nuclear targeting of Php4. The two NLSs are biologically redundant and are sufficient to target a heterologous reporter protein to the nucleus. Under low-iron conditions, a functional GFP-Php4 protein is only partly targeted to the nucleus in imp1Δ and sal3Δ mutant cells. We further found that cells expressing a temperature-sensitive mutation in cut15 exhibit increased cytosolic accumulation of Php4 at the nonpermissive temperature. Further analysis by pull-down experiments revealed that Php4 is a cargo of the karyopherins Imp1, Cut15 and Sal3. Collectively, these results indicate that Php4 can be bound by distinct karyopherins, connecting it into more than one nuclear import pathway.

Nuclear import of human histone lysine-specific demethylase LSD1.

Upregulation and nuclear retention of the human histone demethylase LSD1 are correlated with aggressiveness and poor outcome of several cancer types, but the molecular mechanism of LSD1 nuclear import remains unclear. Here, we found that the N-terminal flexible region of LSD1 contains a nuclear localization signal (NLS), (112)RRKRAK(117). Mutation or deletion of the NLS completely abolished the nuclear import of LSD1, suggesting the motif is a bona fide NLS. More importantly, our GST pull-down assay showed that LSD1 physically interacts with three proteins of importin α family. In addition, our data suggest that the nuclear localization of LSD1 via the NLS is not a cell-type specific event. Thus, these findings demonstrate for the first time that the NLS motif within the N-terminal flexible domain of LSD1 is critical for its nuclear localization via interaction with importin α proteins.

Efficacious inhibition of Importin α/β-mediated nuclear import of human inositol phosphate multikinase

Human inositol phosphate multikinase (IPMK) is a nucleocytoplasmic shuttling protein involved in multiple signal transduction pathways located both in the nucleus and in the cytoplasm. To efficaciously inhibit the conventional nuclear import of IPMK, we first examined the effect of different inhibitors and cellular stressors on nuclear import of enhanced green fluorescent protein monomer and octamer, both fused with a monopartite nuclear localization signal (NLS), in HeLa and H1299 cells. Most efficacious inhibition of conventional nuclear protein import was observed when using Importazole and hydrogen peroxide. Therefore, these substances were then applied to examine nuclear import mechanisms of IPMK. Thereby, we demonstrated that nuclear accumulation of IPMK is significantly lessened, but not abrogated by inhibition of conventional protein import. This indicates that IPMK is imported into the nucleus by both conventional and non-conventional pathways. Furthermore, intracellular distribution of an IPMK mutant with inactivated NLS is unaffected by inhibition of conventional protein import. Obviously, the conventional import of IPMK is entirely mediated by interaction of the Importin α/β heterodimer with IPMK's sole NLS motif (R(320)HRKIYTKKHH). Future research should focus on the hitherto unknown non-conventional import of IPMK and the potential impact of its dysregulation on IPMK signaling pathways regulating cellular growth and proliferation.

Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.

A Masked PY-NLS in Drosophila TIS11 and Its Mammalian Homolog Tristetraprolin

Many RNA-binding proteins (RBPs) dynamically shuttle between the nucleus and the cytoplasm, often exerting different functions in each compartment. Therefore, the nucleo-cytoplasmic distribution of RBPs has a strong impact on their activity. Here we describe the localization and the shuttling properties of the tandem zinc finger RBP dTIS11, which is the Drosophila homolog of mammalian TIS11 proteins. Drosophila and mammalian TIS11 proteins act as destabilizing factors in ARE-mediated decay. At equilibrium, dTIS11 is concentrated mainly in the cytoplasm. We show that dTIS11 is a nucleo-cytoplasmic shuttling protein whose nuclear export is mediated by the exportin CRM1 through the recognition of a nuclear export signal (NES) located in a different region comparatively to its mammalian homologs. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This NLS partially overlaps the second zinc finger ZnF2. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zinc ion unmask dTIS11 and TTP NLS and promote nuclear import. All together, our results indicate that the nuclear export of TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism may rely on a highly conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.

Nuclear Respiratory Factor 2β (NRF-2β) Recruits NRF-2α to the Nucleus by Binding to Importin-α:β via an Unusual Monopartite-Type Nuclear Localization Signal

Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2α, which binds to DNA through its Ets domain, and NRF-2β, which contains the transcription activation domain. The activity of NRF-2 in neurons is regulated by nuclear localization; however, the mechanism by which NRF-2 is imported into the nucleus remains unknown. By using in vitro nuclear import assays and immuno-cytofluorescence, we dissect the nuclear import pathways of NRF-2. We show that both NRF-2α and NRF-2β contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2α and the NLS within NRF-2β (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-α:β. When NRF-2α and NRF-2β form a complex, the nuclear import of NRF-2αβ becomes strictly dependent on the NLS within NRF-2β. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2β NLS contains only two lysine/arginine residues, unlike other known importin-α:β-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-α in almost the same manner and with similar affinity to that of the classical monopartite NLSs, such as c-myc and SV40 T-antigen NLSs. However, the part of the tryptophan array of importin-α that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P3 and the P5 lysine/arginine side chains is not required for the recognition of the NRF-2β NLS. We conclude that the NRF-2β NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS.

CCCTC-binding factor controls its own nuclear transport via regulating the expression of importin 13.

CCCTC-binding factor (CTCF), a multivalent zinc-finger protein, is involved in different aspects of regulation including promoter activation or repression, gene silencing, chromatin insulation, gene imprinting, X-chromosome inactivation, cell growth or differentiation and tumor genesis. However, the molecular mechanisms of CTCF nuclear import remains unclear. In this study, we showed that the expression of CTCF influenced the intracellular distribution of itself, which might go through transport receptor - import 13 (IPO13). We further confirmed that there is a CTCF target site in ipo13 -774∼-573 bp promoter region and CTCF regulates the expression of IPO13. Besides, GST pull-down and Co-IP experiments demonstrated that CTCF interacts with IPO13. Immunofluorescence staining showed that IPO13 influenced intracellular distribution of CTCF. In all, we conclude that CTCF regulates the expression of IPO13, which, in turn, mediates the nuclear import of CTCF.

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1*

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.

IGFBP-2 nuclear translocation is mediated by a functional NLS sequence and is essential for its pro-tumorigenic actions in cancer cells

IGFBP-2 is highly expressed in both the serum and tumor tissues of most cancers, and is considered one of the most significant genes in the signature of major cancers. IGFBP-2 mainly modulates IGF actions in the pericellular space; however, there is considerable evidence to suggest that IGFBP-2 may also act independently of the IGFs. These IGF-independent actions of IGFBP-2 are exerted either via interactions at the cell surface or intracellularly, via interaction with cytoplasmic or nuclear-binding partners. The precise mechanism underlying the intracellular/intranuclear localization of IGFBP-2 remains unclear. In this study, we investigated IGFBP-2 nuclear localization in several common cancer cells with the aim of dissecting the mechanism of its nuclear trafficking. IGFBP-2 is detected in the nuclei of common cancer cells, including breast, prostate and several neuroblastoma cell lines, using cell fractionation and confocal microscopy. Via nuclear import assays, we show that nuclear entry of IGFBP-2 is mediated by the classical nuclear import mechanisms, primarily through importin-α, as demonstrated by the use of blocking, competition and co-immunoprecipitation assays. Bioinformatics analysis of the IGFBP-2 protein sequence with PSORT II identified a classical nuclear localization signal (cNLS) sequence at 179PKKLRPP185, within the IGFBP-2 linker domain, mutagenesis of which abolishes IGFBP-2 nuclear import. Accordingly, the NLSmutIGFBP-2 fails to activate the VEGF promoter, which would otherwise occur in the presence of wild-type IGFBP-2. As a consequence, no activation of angiogenic processes were observed in NLSmutIGFBP-2 expressing SHEP cells when implanted onto our in vivo quail chorio-allantoic membrane model. Taken together, these data show for the first time that IGFBP-2 possesses a functional NLS sequence and that IGFBP-2 actively translocates into the nucleus by a classical nuclear import mechanism, involving formation of IGFBP-2 complexes with importin-α. Nuclear IGFBP-2 is required for the activation of VEGF expression and consequent angiogenesis.

Transcription factor-dependent nuclear localization of a transcriptional repressor in jasmonate hormone signaling

The plant hormone jasmonate (JA) plays an important role in regulating growth, development and immunity. A key step in JA signaling is ligand-dependent assembly of a coreceptor complex consisting of the F-box protein COI1 and JAZ transcriptional repressors. Assembly of this receptor complex results in proteasome-mediated degradation of JAZ repressors, which at resting state bind to and repress the MYC transcription factors. Although the JA receptor complex is believed to function within the nucleus, how this receptor complex enters the nucleus and, more generally, the cell biology of jasmonate signaling are not well understood. In this study, we conducted mutational analysis of the C termini (containing the conserved Jas motif) of two JAZ repressors, JAZ1 and JAZ9. These analyses unexpectedly revealed different subcellular localization patterns of JAZ1ΔJas and JAZ9ΔJas, which were associated with differential interaction of JAZ1ΔJas and JAZ9ΔJas with MYC2 and differential repressor activity in vivo. Importantly, physical interaction with MYC2 appears to play an active role in the nuclear targeting of JAZ1 and JAZ9, and the nuclear localization of JAZ9 was compromised in myc2 mutant plants. We identified a highly conserved arginine residue in the Jas motif that is critical for coupling MYC2 interaction with nuclear localization of JAZ9 and JAZ9 repressor function in vivo. Our results suggest a model for explaining why some JAZΔJas proteins, but not others, confer constitutive JA-insensitivity when overexpressed in plants. Results also provide evidence for a transcription factor-dependent mechanism for nuclear import of a cognate transcriptional repressor JAZ9 in plants.

Yeast importin-β is required for nuclear import of the Mig2 repressor

BackgroundMig2 has been described as a transcriptional factor that in the absence of Mig1 protein is required for glucose repression of the SUC2 gene. Recently it has been reported that Mig2 has two different subcellular localizations. In high-glucose conditions it is a nuclear modulator of several Mig1-regulated genes, but in low-glucose most of the Mig2 protein accumulates in mitochondria. Thus, the Mig2 protein enters and leaves the nucleus in a glucose regulated manner. However, the mechanism by which Mig2 enters into the nucleus was unknown until now.ResultsHere, we report that the Mig2 protein is an import substrate of the carrier Kap95 (importin-β). The Mig2 nuclear import mechanism bypasses the requirement for Kap60 (importin-α) as an adaptor protein, since Mig2 directly binds to Kap95 in the presence of Gsp1(GDP). We also show that the Mig2 nuclear import and the binding of Mig2 with Kap95 are not glucose-dependent processes and require a basic NLS motif, located between lysine-32 and arginine-37. Mig2 interaction with Kap95 was assessed in vitro using purified proteins, demonstrating that importin-β, together with the GTP-binding protein Gsp1, is able to mediate efficient Mig2-Kap95 interaction in the absence of the importin-α (Kap60). It was also demonstrated, that the directionality of Mig2 transport is regulated by association with the small GTPase Gsp1 in the GDP- or GTP-bound forms, which promote cargo recognition and release, respectively.ConclusionsThe Mig2 protein accumulates in the nucleus through a Kap95 and NLS-dependent nuclear import pathway, which is independent of importin-α in Saccharomyces cerevisiae.

Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

A PY-nuclear localization signal is required for nuclear accumulation of HCMV UL79 protein

Human cytomegalovirus UL79 protein is recently reported to be required for transcription or efficient accumulation of late viral mRNAs during viral infection. An absolute nuclear distribution of UL79 proteins has been observed with immunofluorescence assay, both during the infection of Flag-tagged UL79 recombinant virus and in the HFFs expressing HA-tagged UL79, with or without virus infection. However, little is known about the nuclear import mechanism of UL79 protein. Here, by utilizing living cells fluorescent microscopy, a predominant nuclear localization of UL79 protein in living cells was detected. Furthermore, the nuclear import of UL79 protein was demonstrated to be dependent on the transportin-1-mediated pathway. Finally, a hydrophobic PY-nuclear localization signal (PY-NLS) was delineated between the amino acids 66-92 of UL79 protein. Collectively, we provide evidence that a PY-NLS, firstly described in viral proteins, is responsible for the nuclear accumulation of UL79 protein.

APOBEC3B and AID Have Similar Nuclear Import Mechanisms

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.