Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signa...Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal（s） （NLS） has not been identified. KLF8 shares with other KLFs monopartite NLSs （mNLS） and C2H2 zinc fingers （ZFs）, both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn^2＋-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of 〉1.5 ZFs from Cterminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid （aa） 151-200 region almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear localization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.展开更多
For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracyto...For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes （NPC） is mediated by the nuclear transport receptors importin α andβ. Importin α binds to the C-terminus of the capsid protein that comprises a nuclear localization signal （NLS）. The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin α interacts with importin β that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.展开更多
Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mit...Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B （LMB）, a specific inhibitor of Crml, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals （NESs） in pericentrin with diverse export activities. All of the five NESs could bind to Crml in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal （NLS） consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.展开更多
Tip60 is a specific member of MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of nuclear histone acetyltransferases (HAT). It is essential for cellular survival, differentiation, and metabolism. A putative canonical NLS motif ...Tip60 is a specific member of MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of nuclear histone acetyltransferases (HAT). It is essential for cellular survival, differentiation, and metabolism. A putative canonical NLS motif between the chromo domain and the zinc finger of Tip60 was identified. Here we show evidence that Tip60 is associated with importin α as its substrate and transported from cytoplasm to the nucleus. Pull down assay revealed that Tip60 was physically associated with importin α both in vivo and in vitro. Confocal microscopic observation showed that Tip60 and importin α were co-localized with each other. The localization of Tip60 to the nuclear and its interaction with importin α was disrupted when its putative NLS motif for binding to importin α was mutated (219RKRK222 → 219AAAA222). However, attachment of this putative NLS motif to a cytoplasmic protein (YAP 1-210 fragment) promoted its nuclear localization. Based on transient transfection, Tip60 NLS motif mutant showed a substantial reduction in self-acetylation, HAT activity, and apoptotic ability whereas wild type Tip60 did not show such reduction. Taken together, our results demonstrate that importin α transports Tip60 from the cytoplasm to the nucleus through binding to the putative NLS motif of Tip60 for its tumor suppressing function.展开更多
Flowering locus C(FLC)is a central transcriptional repressor that controls flowering time.However,how FLC is imported into the nucleus is unknown.Here,we report that Arabidopsis nucleoporins 62(NUP62),NUP58,and NUP54 ...Flowering locus C(FLC)is a central transcriptional repressor that controls flowering time.However,how FLC is imported into the nucleus is unknown.Here,we report that Arabidopsis nucleoporins 62(NUP62),NUP58,and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importinα-independent manner,via direct interaction.NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel.Importinβsupersensitive to ABA and drought 2(SAD2),a carrier protein,is critical for FLC nuclear import and flower transition,which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex.Proteomics,RNAseq,and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences(NLSs),such as FLC.Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition,and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants.展开更多
Maintenance of cell wall integrity is of great importance not only for plant growth and development,but also for the adaptation of plants to adverse environments.However,how the cell wall integrity is modulated under ...Maintenance of cell wall integrity is of great importance not only for plant growth and development,but also for the adaptation of plants to adverse environments.However,how the cell wall integrity is modulated under salt stress is still poorly understood.Here,we report that a nuclear-localized Agenet domain-containing protein SWO1(SWOLLEN 1)is required for the maintenance of cell wall integrity in Arabidopsis under salt stress.Mutation in SWO1 gene results in swollen root tips,disordered root cell morphology,and root elongation inhibition under salt stress.The swo1 mutant accumulates less cellulose and pectin but more lignin under high salinity.RNA-seq and ChIP-seq assays reveal that SWO1 binds to the promoter of several cell wall-related genes and regulates their expression under saline conditions.Further study indicates that SWO1 interacts with importinɑIMPA1 and IMPA2,which are required for the import of nuclear-localized proteins.The impa1 impa2 double mutant also exhibits root growth inhibition under salt stress and mutations of these two genes aggravate the salt-hypersensitive phenotype of the swo1 mutant.Taken together,our data suggest that SWO1 functions together with importinɑto regulate the expression of cell wall-related genes,which enables plants to maintain cell wall integrity under high salinity.展开更多
文摘Kruppel-like factor 8 （KLF8） transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal（s） （NLS） has not been identified. KLF8 shares with other KLFs monopartite NLSs （mNLS） and C2H2 zinc fingers （ZFs）, both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn^2＋-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of 〉1.5 ZFs from Cterminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid （aa） 151-200 region almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear localization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.
文摘For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes （NPC） is mediated by the nuclear transport receptors importin α andβ. Importin α binds to the C-terminus of the capsid protein that comprises a nuclear localization signal （NLS）. The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin α interacts with importin β that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.
文摘Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B （LMB）, a specific inhibitor of Crml, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals （NESs） in pericentrin with diverse export activities. All of the five NESs could bind to Crml in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal （NLS） consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.
文摘Tip60 is a specific member of MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of nuclear histone acetyltransferases (HAT). It is essential for cellular survival, differentiation, and metabolism. A putative canonical NLS motif between the chromo domain and the zinc finger of Tip60 was identified. Here we show evidence that Tip60 is associated with importin α as its substrate and transported from cytoplasm to the nucleus. Pull down assay revealed that Tip60 was physically associated with importin α both in vivo and in vitro. Confocal microscopic observation showed that Tip60 and importin α were co-localized with each other. The localization of Tip60 to the nuclear and its interaction with importin α was disrupted when its putative NLS motif for binding to importin α was mutated (219RKRK222 → 219AAAA222). However, attachment of this putative NLS motif to a cytoplasmic protein (YAP 1-210 fragment) promoted its nuclear localization. Based on transient transfection, Tip60 NLS motif mutant showed a substantial reduction in self-acetylation, HAT activity, and apoptotic ability whereas wild type Tip60 did not show such reduction. Taken together, our results demonstrate that importin α transports Tip60 from the cytoplasm to the nucleus through binding to the putative NLS motif of Tip60 for its tumor suppressing function.
基金supported by grants from the National Natural Science Foundation(31970730 and 32170721)to H.Hthe Baichuan felowship from College of Life Science and Technology,Huazhong Agricultural University to D.X。
文摘Flowering locus C(FLC)is a central transcriptional repressor that controls flowering time.However,how FLC is imported into the nucleus is unknown.Here,we report that Arabidopsis nucleoporins 62(NUP62),NUP58,and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importinα-independent manner,via direct interaction.NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel.Importinβsupersensitive to ABA and drought 2(SAD2),a carrier protein,is critical for FLC nuclear import and flower transition,which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex.Proteomics,RNAseq,and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences(NLSs),such as FLC.Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition,and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences,Grant XDB27040101(to J-K.Z.)Shanghai Pujiang Program,Grant 20PJ1414800(to C.Z.),and National Natural Science Foundation of China,Grant No.32070295(to C.Z.).
文摘Maintenance of cell wall integrity is of great importance not only for plant growth and development,but also for the adaptation of plants to adverse environments.However,how the cell wall integrity is modulated under salt stress is still poorly understood.Here,we report that a nuclear-localized Agenet domain-containing protein SWO1(SWOLLEN 1)is required for the maintenance of cell wall integrity in Arabidopsis under salt stress.Mutation in SWO1 gene results in swollen root tips,disordered root cell morphology,and root elongation inhibition under salt stress.The swo1 mutant accumulates less cellulose and pectin but more lignin under high salinity.RNA-seq and ChIP-seq assays reveal that SWO1 binds to the promoter of several cell wall-related genes and regulates their expression under saline conditions.Further study indicates that SWO1 interacts with importinɑIMPA1 and IMPA2,which are required for the import of nuclear-localized proteins.The impa1 impa2 double mutant also exhibits root growth inhibition under salt stress and mutations of these two genes aggravate the salt-hypersensitive phenotype of the swo1 mutant.Taken together,our data suggest that SWO1 functions together with importinɑto regulate the expression of cell wall-related genes,which enables plants to maintain cell wall integrity under high salinity.
