Subunit Interactions in the mariner Transposase

Abstract We have studied the Mos1 transposase encoded by the transposable element mariner. This transposase is a member of the “D,D(35)E” superfamily of proteins exhibiting the motif D,D(34)D. It is not known whether this transposase, or other eukaryote transposases manifesting the D,D(35)E domain, functions in a multimeric form. Evidence for oligomerization was found in the negative complementation of Mos1 by an EMS-induced transposase mutation in the catalytic domain. The transposase produced by this mutation has a glycine-to-arginine replacement at position 292. The G292R mutation strongly interferes with the ability of wild-type transposase to catalyze excision of a target element. Negative complementation was also observed for two other EMS mutations, although the effect was weaker than observed with G292R. Results from the yeast two-hybrid system also imply that Mos1 subunits interact, suggesting the possibility of subunit oligomerization in the transposition reaction. Overproduction of Mos1 subunits through an hsp70 promoter also inhibits excision of the target element, possibly through autoregulatory feedback on transcription or through formation of inactive or less active oligomers. The effects of both negative complementation and overproduction may contribute to the regulation of mariner transposition.

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Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

Journal of Biological Chemistry ◽

10.1074/jbc.m009810200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11980-11987 ◽

Cited By ~ 88

Author(s):

Steven A. Haney ◽

Elizabeth Glasfeld ◽

Cynthia Hale ◽

David Keeney ◽

Zhizhen He ◽

...

Keyword(s):

Hybrid System ◽

Enzyme Linked Immunosorbent Assay ◽

Wild Type ◽

Yeast Two Hybrid ◽

Conserved Sequence ◽

Yeast Two Hybrid System ◽

C Terminus ◽

Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

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PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system.

The Journal of Cell Biology ◽

10.1083/jcb.128.3.263 ◽

1995 ◽

Vol 128 (3) ◽

pp. 263-271 ◽

Cited By ~ 211

Author(s):

J Staudinger ◽

J Zhou ◽

R Burgess ◽

S J Elledge ◽

E N Olson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Hybrid System ◽

Catalytic Domain ◽

Yeast Two Hybrid ◽

Kinase C ◽

Yeast Two Hybrid System ◽

Wide Range ◽

Two Hybrid

Protein kinase C (PKC) plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors. Upon activation by diacylglycerol, PKC translocates to different subcellular sites where it phosphorylates numerous proteins, most of which are unidentified. We used the yeast two-hybrid system to identify proteins that interact with activated PKC alpha. Using the catalytic region of PKC fused to the DNA binding domain of yeast GAL4 as "bait" to screen a mouse T cell cDNA library in which cDNA was fused to the GAL4 activation domain, we cloned several novel proteins that interact with C-kinase (PICKs). One of these proteins, designated PICK1, interacts specifically with the catalytic domain of PKC and is an efficient substrate for phosphorylation by PKC in vitro and in vivo. PICK1 is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK1 and other PICKs may play important roles in mediating the actions of PKC.

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Role of Adaptor Complex AP-3 in Targeting Wild-Type and Mutated CD63 to Lysosomes

Molecular Biology of the Cell ◽

10.1091/mbc.01-08-0409 ◽

2002 ◽

Vol 13 (3) ◽

pp. 1071-1082 ◽

Cited By ~ 160

Author(s):

Brian A. Rous ◽

Barbara J. Reaves ◽

Gudrun Ihrke ◽

John A.G. Briggs ◽

Sally R. Gray ◽

...

Keyword(s):

Hybrid System ◽

Rat Kidney ◽

Wild Type ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Strength Of Interaction ◽

Early Endosomes ◽

Genes Encoding ◽

Normal Rat ◽

Two Hybrid

CD63 is a lysosomal membrane protein that belongs to the tetraspanin family. Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM. Strong, tyrosine-dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit μ3 was observed using a yeast two-hybrid system. The strength of interaction of mutated tail sequences with μ3 correlated with the degree of lysosomal localization of similarly mutated human CD63 molecules in stably transfected normal rat kidney cells. Mutated CD63 containing the cytosolic tail sequence GYEVI, which interacted strongly with μ3 but not at all with μ2 in the yeast two-hybrid system, localized to lysosomes in transfected normal rat kidney and NIH-3T3 cells. In contrast, it localized to the cell surface in transfected cells ofpearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the δ subunit of AP-3. Thus, AP-3 is absolutely required for the delivery of this mutated CD63 to lysosomes. Using this AP-3–dependent mutant of CD63, we have shown that AP-3 functions in membrane traffic from thetrans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes.

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Functional Analyses of the Pto Resistance Gene Family in Tomato and the Identification of a Minor Resistance Determinant in a Susceptible Haplotype

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.3.281 ◽

2002 ◽

Vol 15 (3) ◽

pp. 281-291 ◽

Cited By ~ 54

Author(s):

Jeff H. Chang ◽

Yin-Shan Tai ◽

Adriana J. Bernal ◽

Daniel T. Lavelle ◽

Brian J. Staskawicz ◽

...

Keyword(s):

Cell Death ◽

Amino Acid ◽

Hybrid System ◽

Dependent Manner ◽

Wild Type ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

A Minor ◽

Two Hybrid

Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv. tomato expressing avrPto. The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4,) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases. In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system. The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophos-phorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf. LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction. The interaction with AvrPto was unique to Pto in the yeast two-hybrid system. LescPth2 autophosphorylated in vitro as a fusion protein. LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro. Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana. LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf. Consequently, some homologs could potentially encode functional resistance proteins. LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a ‘susceptible’ haplotype encoding a minor recognition determinant.

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Genetic Analysis of nif Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a NifL-NifA Complex That Is Implicated in Regulated Expression of nif Genes

Journal of Bacteriology ◽

10.1128/jb.181.20.6535-6539.1999 ◽

1999 ◽

Vol 181 (20) ◽

pp. 6535-6539 ◽

Cited By ~ 29

Author(s):

Shi Lei ◽

Lakshmidevi Pulakat ◽

Narasaiah Gavini

Keyword(s):

Dna Binding ◽

Hybrid System ◽

Transcriptional Activation ◽

Catalytic Domain ◽

Dna Binding Domain ◽

Yeast Two Hybrid ◽

Activation Domain ◽

Yeast Two Hybrid System ◽

Dna Fragment ◽

Two Hybrid

ABSTRACT In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes, nifL and nifA, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O2 tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of nifgenes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of nifL was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of nifA was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression oflacZ and his3 genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA.

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