Evidence for Interaction of Schizophyllum commune Y Mating-Type Proteins in Vivo

Abstract The Aα mating-type locus of Schizophyllum commune regulates sexual development and contains the code for two proteins, Y and Z, which are thought to form a complex and function as a transcription factor. Import of these proteins into the nucleus may be an essential step in Aα-regulated sexual development. The Y proteins contain a bipartite basic sequence, which is an excellent candidate for a nuclear localization sequence (NLS), while Z proteins contain no such sequence. Here we describe experiments in which deletions were made in the putative NLS sequence of Y4. We show that this putative NLS is essential to the function of the Y protein and capable of mislocalizing green fluorescent protein (GFP) to the nucleus in Saccharomyces cerevisiae. Further, we describe genetic experiments that demonstrate the first Y-Y protein interactions in vivo. These results are consistent with our previously postulated hypothesis that the Y-Z complex is likely to be of a higher order than dimer.

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Nuclear Import of Histone H2a and H2b Is Mediated by a Network of Karyopherins

The Journal of Cell Biology ◽

10.1083/jcb.153.2.251 ◽

2001 ◽

Vol 153 (2) ◽

pp. 251-262 ◽

Cited By ~ 113

Author(s):

Nima Mosammaparast ◽

Kelley R. Jackson ◽

Yurong Guo ◽

Cynthia J. Brame ◽

Jeffrey Shabanowitz ◽

...

Keyword(s):

Nuclear Import ◽

Fluorescent Protein ◽

Nuclear Localization Sequence ◽

Temperature Sensitive ◽

Histone H2a ◽

Amino Terminal ◽

Core Histones ◽

Localization Sequence ◽

Green Fluorescent

The first step in the assembly of new chromatin is the cell cycle–regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

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Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

International Journal of Molecular Sciences ◽

10.3390/ijms21041414 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1414 ◽

Cited By ~ 5

Author(s):

Hui Li ◽

Xiaobao Ying ◽

Lina Shang ◽

Bryce Redfern ◽

Nicholas Kypraios ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Nuclear Localization Sequence ◽

Localization Sequence ◽

Candidatus Liberibacter ◽

Phloem Necrosis ◽

Liberibacter Asiaticus ◽

Green Fluorescent

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

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Nuclear targeting of the β isoform of Type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its α-helix 7

Biochemical Journal ◽

10.1042/bj3460587 ◽

2000 ◽

Vol 346 (3) ◽

pp. 587-591 ◽

Cited By ~ 50

Author(s):

Antonio CIRUELA ◽

Katherine A. HINCHLIFFE ◽

Nullin DIVECHA ◽

Robin F. IRVINE

Keyword(s):

Nuclear Localization ◽

Fluorescent Protein ◽

Physiological Role ◽

Nuclear Localization Sequence ◽

Type Ii ◽

Nuclear Targeting ◽

Phosphatidylinositol Phosphate ◽

Localization Sequence ◽

Α Helix ◽

Green Fluorescent

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIβ isoform. Type IIβ PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the α (cytosolic) and β PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of α-helix (α-helix 7) that is specific to the β isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIβ PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

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In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Biomolecules ◽

10.3390/biom10010142 ◽

2020 ◽

Vol 10 (1) ◽

pp. 142 ◽

Cited By ~ 1

Author(s):

Arman Kulyyassov ◽

Vasily Ogryzko

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Fluorescent Protein ◽

Quantitative Estimation ◽

Multiple Reaction Monitoring ◽

Negative Control ◽

Protein Protein Interactions ◽

Close Proximity ◽

Green Fluorescent

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

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Splicing signals and factors in plant intron removal

Biochemical Society Transactions ◽

10.1042/bst0300146 ◽

2002 ◽

Vol 30 (2) ◽

pp. 146-149 ◽

Cited By ~ 13

Author(s):

J. W. S. Brown ◽

C. G. Simpson ◽

G. Thow ◽

G. P. Clark ◽

S. N. Jennings ◽

...

Keyword(s):

Protein Interactions ◽

Viral Vectors ◽

Binding Proteins ◽

Fluorescent Protein ◽

Splicing Factors ◽

Small Nuclear Rna ◽

Intron Removal ◽

Arabidopsis Proteins ◽

Green Fluorescent

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

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Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.01701-08 ◽

2009 ◽

Vol 191 (9) ◽

pp. 3149-3161 ◽

Cited By ~ 56

Author(s):

Suzanne R. Lybarger ◽

Tanya L. Johnson ◽

Miranda D. Gray ◽

Aleksandra E. Sikora ◽

Maria Sandkvist

Keyword(s):

Vibrio Cholerae ◽

Protein Interactions ◽

Fluorescent Protein ◽

Outer Membrane Proteins ◽

Subcellular Location ◽

Type Ii Secretion ◽

Type Ii ◽

Protein Fusion ◽

Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

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Degradation of SAMHD1 by Vpx Is Independent of Uncoating

Journal of Virology ◽

10.1128/jvi.03575-14 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5701-5713 ◽

Cited By ~ 7

Author(s):

Paula Jáuregui ◽

Eric C. Logue ◽

Megan L. Schultz ◽

Stephanie Fung ◽

Nathaniel R. Landau

Keyword(s):

T Cells ◽

Green Fluorescent Protein ◽

Fusion Protein ◽

Hela Cell ◽

Nuclear Localization ◽

Fluorescent Protein ◽

Nuclear Localization Sequence ◽

Hela Cell Lines ◽

Localization Sequence ◽

Green Fluorescent

ABSTRACTSterile alpha motif domain and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid and resting T cells. Lentiviruses such as HIV-2 and some simian immunodeficiency viruses (SIVs) counteract the restriction by encoding Vpx or Vpr, accessory proteins that are packaged in virions and which, upon entry of the virus into the cytoplasm, induce the proteasomal degradation of SAMHD1. As a tool to study these mechanisms, we generated HeLa cell lines that express a fusion protein termed NLS.GFP.SAM595 in which the Vpx binding domain of SAMHD1 is fused to the carboxy terminus of green fluorescent protein (GFP) and a nuclear localization signal is fused to the amino terminus of GFP. Upon incubation of Vpx-containing virions with the cells, the NLS.GFP.SAM595 fusion protein was degraded over several hours and the levels remained low over 5 days as the result of continued targeting of the CRL4 E3 ubiquitin ligase. Degradation of the fusion protein required that it contain a nuclear localization sequence. Fusion to the cytoplasmic protein muNS rendered the protein resistant to Vpx-mediated degradation, confirming that SAMHD1 is targeted in the nucleus. Virions treated with protease inhibitors failed to release Vpx, indicating that Gag processing was required for Vpx release from the virion. Mutations in the capsid protein that altered the kinetics of virus uncoating and the Gag binding drug PF74 had no effect on the Vpx-mediated degradation. These results suggest that Vpx is released from virions without a need for uncoating of the capsid, allowing Vpx to transit to the nucleus rapidly upon entry into the cytoplasm.IMPORTANCESAMHD1 restricts lentiviral replication in myeloid cells and resting T cells. Its importance is highlighted by the fact that viruses such as HIV-2 encode an accessory protein that is packaged in the virion and is dedicated to inducing SAMHD1 degradation. Vpx needs to act rapidly upon infection to allow reverse transcription to proceed. The limited number of Vpx molecules in a virion also needs to clear the cell of SAMHD1 over a prolonged period of time. Using an engineered HeLa cell line that expresses a green fluorescent protein (GFP)-SAMHD1 fusion protein, we showed that the Vpx-dependent degradation occurs without a need for viral capsid uncoating. In addition, the fusion protein was degraded only when it was localized to the nucleus, confirming that SAMHD1 is targeted in the nucleus and thus explaining why Vpx also localizes to the nucleus.

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Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

The Journal of Cell Biology ◽

10.1083/jcb.200911091 ◽

2010 ◽

Vol 189 (4) ◽

pp. 739-754 ◽

Cited By ~ 302

Author(s):

Nina C. Hubner ◽

Alexander W. Bird ◽

Jürgen Cox ◽

Bianca Splettstoesser ◽

Peter Bandilla ◽

...

Keyword(s):

Protein Interactions ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Small Scale ◽

Mitotic Spindles ◽

Cellular Processes ◽

Green Fluorescent ◽

Highly Sensitive Detection ◽

Interaction Proteomics

Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.

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Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.6122 ◽

1997 ◽

Vol 17 (10) ◽

pp. 6122-6130 ◽

Cited By ~ 119

Author(s):

A W Johnson

Keyword(s):

Fluorescent Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Nuclear Localization Sequence ◽

Wild Type ◽

Gene Encoding ◽

Cytoplasmic Rna ◽

Localization Sequence ◽

Green Fluorescent

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.

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Transcriptome and Functional Analysis of Mating in the Basidiomycete Schizophyllum commune

Eukaryotic Cell ◽

10.1128/ec.05214-11 ◽

2011 ◽

Vol 11 (5) ◽

pp. 571-589 ◽

Cited By ~ 27

Author(s):

Susann Erdmann ◽

Daniela Freihorst ◽

Marjatta Raudaskoski ◽

Wolfgang Schmidt-Heck ◽

Elke-Martina Jung ◽

...

Keyword(s):

Mating Type ◽

Fluorescent Protein ◽

Expression Patterns ◽

Functional Characterization ◽

Schizophyllum Commune ◽

Northern Hybridization ◽

Cell Periphery ◽

Dependent Manner ◽

Type Locus ◽

Mating Type Locus

ABSTRACTIn this study, we undertook a functional characterization and transcriptome analysis that enabled a comprehensive study of the mating type loci of the mushroomSchizophyllum commune. Induced expression of both thebar2receptor and thebap2(2)pheromone gene within 6 to 12 h after mates' contact was demonstrated by quantitative real-time PCR. Similar temporal expression patterns were confirmed for the allelicbbr1receptor andbbp1pheromone-encoding genes by Northern hybridization. Interestingly, the fusion of clamp connections to the subterminal cell was delayed in mating interactions in which one of the compatible partners expressed thebar2receptor with a truncated C terminus. This developmental delay allowed the visualization of a green fluorescent protein (Gfp)-labeled truncated receptor at the cell periphery, consistent with a localization in the plasma membrane of unfused pseudoclamps. This finding does not support hypotheses envisioning a receptor localization to the nuclear membrane facilitating recognition between the two different nuclei present in each dikaryotic cell. Rather, Gfp fluorescence observed in such pseudoclamps indicated a role of receptor-pheromone interaction in clamp fusion. Transcriptome changes associated with mating interactions were analyzed in order to identify a role for pheromone-receptor interactions. We detected a total of 89 genes that were transcriptionally regulated in a mating type locusA-dependent manner, employing a cutoff of 5-fold changes in transcript abundance. Upregulation in cell cycle-related genes and downregulation of genes involved in metabolism were seen with this set of experiments. In contrast, mating type locusB-dependent transcriptome changes were observed in 208 genes, with a specific impact on genes related to cell wall and membrane metabolism, stress response, and the redox status of the cell.

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