Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

Genetic Analysis of Suppressors of the Vea1 Mutation in Aspergillus Nidulans

Light-dependent conidiation in the filamentous ascomycete, Aspergillus nidulans, is contingent on the allelic state of the velvet (veA) gene. Light dependence is abolished by a mutation in this gene (veA1), which allows conidiation to occur in the absence of light. We have isolated and characterized six extragenic suppressors of veA1 that restore the light-dependent conidiation phenotype. Alleles of four genes, defined by complementation tests, were subjected to extensive genetic and phenotypic analysis. The results of light-dark shifting experiments and the phenotypes of double mutant combinations are consistent with the possibility that the expression of the light-dependent phenotype is regulated by specific interactions of the suppressor gene products with the velvet gene product and with each other.     

VEA1 is required for cleistothecial formation and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a pathogenic fungus dependent on dimorphism for virulence. Among the four described Velvet family genes, two of them, Ryp2 and Ryp3, have been shown to be required for dimorphism. It is known that Velvet A (VeA) is necessary for sexual development and toxin production in Aspergillus nidulans. However, the role of the VeA ortholog in H. capsulatum has not yet been explored. Vea1, H. capsulatum homolog of VeA, was studied to determine its role in cleistothecial formation, dimorphism, and virulence. H. capsulatum Vea1 restores cleistothecial formation and partially restores sterigmatocystin production in an A. nidulans veA deletion strain. Furthermore, silencing VEA1 in an H. capsulatum strain capable of forming cleistothecia abolishes cleistothecial formation. Silenced strains also switch to mycelial phase faster, and show impaired switching to the yeast phase once in mycelial phase. Virulence in mice and macrophages is attenuated in VEA1 silenced strains and silenced strains demonstrate increased sensitivity during growth under acidic conditions. These results indicate that H. capsulatum Vea1 shares a similar role in development as VeA. H. capsulatum is also more susceptible to growth in acidic conditions when VEA1 is silenced, which may contribute to the silenced strains' attenuated virulence in mice and macrophages.     

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

Large subunit of translation initiation factor--3 p170 contains potentially functional nuclear localization signals

Eukaryotic translation factors and their subunits can have independent cellular functions, including regulation of nuclear events. We analyzed primary structure of p170 large subunit of human translation initiation factor eIF3 and found four potential bipartite nuclear localization signals (NLS). Then we studied whether these NLS were functional, that is were able to direct protein to cell nucleus. Complementary DNA of p170 fragments were expressed in cultured CV-1 and Cos-1 green monkey cells, and localization of fused with GFP proteins was determined by fluorescent microscopy. We established that p170 molecule possessed at least two functional NLS which determined nuclear localization of p170 fragments. At the same time more long p170 fragments containing the same functional NLS could be retained in cytoplasm. We speculate that either using specific factors or after limited proteolysis p170 can enter cell nucleus and participate in genome expression regulation. Also we do not exclude the possibility that functioning of p170 in cytoplasm can be regulated by reversible binding of importins to its NLS.     

The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.     

Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals.

Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.     

Identification and characterization of nuclear localization signals within the nucleocapsid protein VP15 of white spot syndrome virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.     

BRD7核定位信号的结构分析和功能鉴定

目的:对BRD7的核定位信号进行预测、结构分析和功能鉴定,并考察其对BRD7亚细胞定位的影响。方法:通过生物信息学对BRD7的核定位信号进行预测和结构分析,然后利用绿色荧光蛋白(GFP)介导的直接荧光和间接免疫荧光定位方法分别对核定位信号的功能进行鉴定,并考察其对BRD7亚细胞定位的影响。结果:BRD7的65～96位氨基酸残基具有潜在核定位信号(NLS)的结构特征,该核定位信号包含3簇碱性氨基酸残基,可视为由2个紧密相邻、部分重叠的双向核靶序列NLS1和NLS2组成;并发现NLS及其构成上的NLS1和NLS2均具有介导异源蛋白GFP胞核定位的功能,从而证实BRD7的65～96位残基为BRD7功能性核定位信号所在区域,且单簇碱性氨基酸残基的缺失不足以破坏其核定位信号的功能;同时发现野生型BRD7呈胞核分布,而核定位信号缺失型BRD7主要呈胞浆分布。结论:BRD7的65～96位氨基酸残基为BRD7功能性核定位信号所在区域,在BRD7胞核分布模式中发挥了十分重要的作用。     

The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.     

Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans

Light sensing is very important for organisms in all biological kingdoms to adapt to changing environmental conditions. It was discovered recently that plant-like phytochrome is involved in light sensing in the filamentous fungus Aspergillus nidulans[1]. Here, we show that phytochrome (FphA) is part of a protein complex containing LreA (WC-1) and LreB (WC-2) [2, 3], two central components of the Neurospora crassa blue-light-sensing system. We found that FphA represses sexual development and mycotoxin formation, whereas LreA and LreB stimulate both. Surprisingly, FphA interacted with LreB and with VeA, another regulator involved in light sensing and mycotoxin biosynthesis. LreB also interacted with LreA. All protein interactions occurred in the nucleus, despite cytoplasmic subfractions of the proteins. Whereas the FphA-VeA interaction was dependent on the presence of the linear tetrapyrrole in FphA, the interaction between FphA and LreB was chromophore independent. These results suggest that morphological and physiological differentiations in A. nidulans are mediated through a network consisting of FphA, LreA, LreB, and VeA acting in a large protein complex in the nucleus, sensing red and blue light.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp.

Aspergillus spp. are frequently occurring seed-colonizing fungi that complete their disease cycles through the development of asexual spores, which function as inocula, and through the formation of cleistothecia and sclerotia. We found that development of all three of these structures in Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus is affected by linoleic acid and light. The specific morphological effects of linoleic acid include induction of precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotium production in some A. flavus strains in which sclerotium production decreases in the light but increases in the dark. In A. nidulans, both asexual spore production and sexual spore production were altered by linoleic acid. Spore development was induced in all three species by hydroperoxylinoleic acids, which are linoleic acid derivatives that are produced during fungal colonization of seeds. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light treatments also significantly increased asexual spore production in all three species. The sporogenic effects of light, linoleic acid, and linoleic acid derivatives on A. nidulans required an intact veA gene. The sporogenic effects of light and linoleic acid on Aspergillus spp., as well as members of other fungal genera, suggest that these factors may be significant environmental signals for fungal development.