scholarly journals Maize defective kernel mutant generated by insertion of a Ds element in a gene encoding a highly conserved TTI2 cochaperone

2017 ◽  
Vol 114 (20) ◽  
pp. 5165-5170 ◽  
Author(s):  
Nelson Garcia ◽  
Yubin Li ◽  
Hugo K. Dooner ◽  
Joachim Messing
Keyword(s):  
Telomere Maintenance ◽  
Phosphatidylinositol 3 Kinase ◽  
Yeast Two Hybrid ◽  
Gene Encoding ◽  
Interacting Protein ◽  
T Complex ◽  
Embryonic Lethal ◽  
Autonomous Element ◽  
Damage Response ◽  
Two Hybrid

We have used the newly engineered transposable element Dsg to tag a gene that gives rise to a defective kernel (dek) phenotype. Dsg requires the autonomous element Ac for transposition. Upon excision, it leaves a short DNA footprint that can create in-frame and frameshift insertions in coding sequences. Therefore, we could create alleles of the tagged gene that confirmed causation of the dek phenotype by the Dsg insertion. The mutation, designated dek38-Dsg, is embryonic lethal, has a defective basal endosperm transfer (BETL) layer, and results in a smaller seed with highly underdeveloped endosperm. The maize dek38 gene encodes a TTI2 (Tel2-interacting protein 2) molecular cochaperone. In yeast and mammals, TTI2 associates with two other cochaperones, TEL2 (Telomere maintenance 2) and TTI1 (Tel2-interacting protein 1), to form the triple T complex that regulates DNA damage response. Therefore, we cloned the maize Tel2 and Tti1 homologs and showed that TEL2 can interact with both TTI1 and TTI2 in yeast two-hybrid assays. The three proteins regulate the cellular levels of phosphatidylinositol 3-kinase-related kinases (PIKKs) and localize to the cytoplasm and the nucleus, consistent with known subcellular locations of PIKKs. dek38-Dsg displays reduced pollen transmission, indicating TTI2’s importance in male reproductive cell development.

scholarly journals UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Min Ji Yoon ◽  
Boyoon Choi ◽  
Eun Jin Kim ◽  
Jiyeon Ohk ◽  
Chansik Yang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Molecular Chaperones ◽  
Ectopic Expression ◽  
Protein Aggregates ◽  
Yeast Two Hybrid ◽  
Ubiquitinated Protein ◽  
Interacting Protein ◽  
Autophagy Pathway ◽  
Cooperative Relationship ◽  
Two Hybrid

Abstractp62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

HZwint-1, a novel human kinetochore component that interacts with HZW10

2000 ◽  
Vol 113 (11) ◽  
pp. 1939-1950 ◽  
Author(s):  
D.A. Starr ◽  
R. Saffery ◽  
Z. Li ◽  
A.E. Simpson ◽  
K.H. Choo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Dicentric Chromosomes ◽  
Centromere Function ◽  
Coiled Coil Domain ◽  
Gfp Fluorescence ◽  
Two Hybrid ◽  
Active Centromere

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

scholarly journals Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

1999 ◽  
Vol 144 (3) ◽  
pp. 403-411 ◽  
Author(s):  
Shun'ichi Kuroda ◽  
Noritaka Nakagawa ◽  
Chiharu Tokunaga ◽  
Kenji Tatematsu ◽  
Katsuyuki Tanizawa
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Rat Brain ◽  
Axonal Outgrowth ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Kinase C ◽  
Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3

2008 ◽  
Vol 389 (10) ◽  
Author(s):  
Hye Jin Yun ◽  
Jungsun Kwon ◽  
Wongi Seol
Keyword(s):  
Transcriptional Activity ◽  
Constitutive Androstane Receptor ◽  
Splicing Factor ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Functional Studies ◽  
Reporter Activity ◽  
Inhibitory Function ◽  
Two Hybrid ◽  
Hybrid Screening

Abstract The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

Abstract 3600: HspA12B Promotes Angiogenesis through Suppressing AKAP12 and Up-Regulating VEGF Pathway

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Rebecca J Steagall ◽  
Fang Hua ◽  
Mahesh Thirunazukarasu ◽  
Lijun Zhan ◽  
Chuanfu Li ◽  
...  
Keyword(s):  
Cancer Metastasis ◽  
Yeast Two Hybrid ◽  
Vegf Expression ◽  
Tubule Formation ◽  
Interacting Protein ◽  
Over Expression ◽  
Vegf Pathway ◽  
Two Hybrid ◽  
Hybrid Screening

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis in vitro . The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.

scholarly journals Requirement for Bbp1p in the Proper Mitotic Functions of Cdc5p in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (4) ◽  
pp. 1711-1723 ◽  
Author(s):  
Chong J. Park ◽  
Sukgil Song ◽  
Thomas H. Giddings ◽  
Hyeon-Su Ro ◽  
Krisada Sakchaisri ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Spindle Pole ◽  
Stable Complex ◽  
Yeast Two Hybrid ◽  
Binding Studies ◽  
Interacting Protein ◽  
Spindle Pole Bodies ◽  
Vitro Binding ◽  
Two Hybrid

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pΔC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p243–385), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pΔC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5ΔC-BBP1243–385 under CDC5 promoter control partially complemented the cdc5Δ defect. These data suggest that Bbp1pΔC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

scholarly journals Identification of a Plum pox virus CI-Interacting Protein from Chloroplast That Has a Negative Effect in Virus Infection

2006 ◽  
Vol 19 (3) ◽  
pp. 350-358 ◽  
Author(s):  
I. Jiménez ◽  
L. López ◽  
J. M. Alamillo ◽  
A. Valli ◽  
J. A. García
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Movement ◽  
Host Factors ◽  
Plum Pox Virus ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Yeast Two Hybrid System ◽  
Negative Effect ◽  
Virus Interactions ◽  
Two Hybrid

The cylindrical inclusion (CI) protein of potyviruses is involved in virus replication and cell-to-cell movement. These two processes should rely on multiple plant-virus interactions; however, little is known about the host factors that are involved in, or that may interfere with, CI functions. By using a yeast two-hybrid system, the CI protein from Plum pox virus (PPV) was found to interact with the photosystem I PSI-K protein, the product of the gene psaK, of Nicotiana benthamiana. Coexpression of PPV CI was shown to cause a decrease in the accumulation level of PSI-K transiently expressed in N. benthamiana leaves. To test the biological relevance of this interaction, we have analyzed the infection of PPV in N. benthamiana plants in which psaK gene expression has been silenced by RNA interference, as well as in Arabidopsis thaliana psaK knockout plants. Our results show that downregulation of the psaK gene leads to higher PPV accumulation, suggesting a role for the CI-PSI-K interaction in PPV infection.

Using the yeast two-hybrid assay to discover protein partners for the leucine-rich amelogenin peptide and for tuftelin-interacting protein 11

2006 ◽  
Vol 114 (s1) ◽  
pp. 276-279 ◽  
Author(s):  
Hong-Jun Wang ◽  
Sissada Tannukit ◽  
Xin Wen ◽  
Jason L. Shapiro ◽  
Malcolm L. Snead ◽  
...  
Keyword(s):  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Protein Partners ◽  
Two Hybrid
Sign in / Sign up

Export Citation Format

Share Document