Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.

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The 30-kD Domain of Protein 4.1 Mediates Its Binding to the Carboxyl Terminus of pICln, a Protein Involved in Cellular Volume Regulation

Blood ◽

10.1182/blood.v92.4.1442 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1442-1447 ◽

Cited By ~ 26

Author(s):

Chieh-Ju C. Tang ◽

Tang K. Tang

Keyword(s):

Hybrid System ◽

Volume Regulation ◽

Carboxyl Terminus ◽

Cdna Clones ◽

Yeast Two Hybrid ◽

Protein 4.1 ◽

Yeast Two Hybrid System ◽

Cellular Volume ◽

Direct Association ◽

Two Hybrid

Abstract Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione–labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. © 1998 by The American Society of Hematology.

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Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System

Journal of Bacteriology ◽

10.1128/jb.185.8.2393-2401.2003 ◽

2003 ◽

Vol 185 (8) ◽

pp. 2393-2401 ◽

Cited By ~ 11

Author(s):

Yi-Ying Lee ◽

Chiung-Fang Chang ◽

Chueh-Ling Kuo ◽

Meng-Ching Chen ◽

Chien Hung Yu ◽

...

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Protein Interactions ◽

Large Subunit ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

ABSTRACT The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpYΔI) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

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Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/0005-2728(96)00013-8 ◽

1996 ◽

Vol 1274 (1-2) ◽

pp. 67-72 ◽

Cited By ~ 5

Author(s):

Chie Moritani ◽

Ken Sawada ◽

Kouji Takemoto ◽

Yongchol Shin ◽

Shingo Nemoto ◽

...

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Yeast Two Hybrid ◽

F1 Atpase ◽

Yeast Two Hybrid System ◽

Atpase Subunits ◽

Two Hybrid

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Identification and analysis of Escherichia coli proteins that interact with the histidine kinase NtrB in a yeast two-hybrid system

Molecular Genetics and Genomics ◽

10.1007/s00438-003-0866-7 ◽

2003 ◽

Vol 269 (4) ◽

pp. 574-581 ◽

Cited By ~ 5

Author(s):

P. Salinas ◽

A. Contreras

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Histidine Kinase ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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PII T-Loop Mutations Affecting Signal Transduction to NtrB Also Abolish Yeast Two-Hybrid Interactions

Journal of Bacteriology ◽

10.1128/jb.184.13.3746-3748.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3746-3748 ◽

Cited By ~ 18

Author(s):

Isabel Martínez-Argudo ◽

Asunción Contreras

Keyword(s):

Escherichia Coli ◽

Signal Transduction ◽

Hybrid System ◽

Yeast Two Hybrid ◽

Regulatory Interactions ◽

Pii Protein ◽

Yeast Two Hybrid System ◽

Two Component ◽

Pii Proteins ◽

Two Hybrid

ABSTRACT Mutations A49P and Δ47-53 at the T loop of the Escherichia coli GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schepke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment. The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins.

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Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System

Journal of Bacteriology ◽

10.1128/jb.186.7.2224.2004 ◽

2004 ◽

Vol 186 (7) ◽

pp. 2224-2224

Author(s):

Yi-Ying Lee ◽

Chiung-Fang Chang ◽

Chueh-Ling Kuo ◽

Meng-Ching Chen ◽

Chien Hung Yu ◽

...

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Protein Interactions ◽

Substrate Recognition ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

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Study of the Interaction between Bacteriophage T4 asiA and Escherichia coli ς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity to E. coli Cells by Coexpression of a Truncated ς70 Fragment

Journal of Bacteriology ◽

10.1128/jb.181.18.5855-5859.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5855-5859 ◽

Cited By ~ 19

Author(s):

Umender K. Sharma ◽

Sudha Ravishankar ◽

Radha Krishan Shandil ◽

P. V. K. Praveen ◽

T. S. Balganesh

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Bacteriophage T4 ◽

Amino Acid Residues ◽

Yeast Two Hybrid ◽

E Coli ◽

Yeast Two Hybrid System ◽

C Terminus ◽

T4 Phage ◽

Two Hybrid

ABSTRACT The interaction of T4 phage-encoded anti-sigma factor, asiA, andEscherichia coli ς70 was studied by using the yeast two-hybrid system. Truncation of ς70 to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased β-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated ς70 fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated ς70 and asiA could be expressed in large quantities and purified.

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The 30-kD Domain of Protein 4.1 Mediates Its Binding to the Carboxyl Terminus of pICln, a Protein Involved in Cellular Volume Regulation

Blood ◽

10.1182/blood.v92.4.1442.416k14_1442_1447 ◽

1998 ◽

Vol 92 (4) ◽

pp. 1442-1447 ◽

Cited By ~ 2

Author(s):

Chieh-Ju C. Tang ◽

Tang K. Tang

Keyword(s):

Hybrid System ◽

Volume Regulation ◽

Carboxyl Terminus ◽

Cdna Clones ◽

Yeast Two Hybrid ◽

Protein 4.1 ◽

Yeast Two Hybrid System ◽

Cellular Volume ◽

Direct Association ◽

Two Hybrid

Erythrocyte protein 4.1 (P4.1) is an 80-kD cytoskeletal protein that is important for the maintenance of the structural integrity and flexibility of the red blood cell membrane. Limited chymotryptic digestion of erythroid P4.1 yields 4 structural domains corresponding to the 30-, 16-, 10-, and 22/24-kD domains. Using a yeast two-hybrid system, we isolated cDNA clones encoding pICln that specifically interacts with the 30-kD domain of P4.1. In this report, we show that the carboxyl-terminus (amino acid residues 103-237) of pICln binds to the 30-kD domain of P4.1 in a yeast two-hybrid system. The direct association between the 30-kD domain of P4.1 and pICln was further confirmed by the following findings: (1) the S35-methione–labeled pICln specifically bound to both GST/P4.1-80 (80 kD) and GST/P4.1-30 (30 kD) fusion proteins, but not to the proteins that lack the 30-kD domain; (2) coimmunoprecipitation analysis of the cell extracts from transfected SiHa cells showed that pICln and P4.1 associate in transfected cells. It was reported that pICln can form a complex with actin and may play a role involved in cellular volume regulation. The direct association between P4.1 and pICln suggests that pICln may link P4.1-bound cytoskeletal elements to an unidentified volume-sensitive chloride channel. © 1998 by The American Society of Hematology.

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Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli

Microbiology ◽

10.1099/mic.0.26355-0 ◽

2003 ◽

Vol 149 (8) ◽

pp. 2093-2106 ◽

Cited By ~ 72

Author(s):

Elizabeth A. Creasey ◽

Robin M. Delahay ◽

Sarah J. Daniell ◽

Gad Frankel

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Type Iii Secretion ◽

Effector Proteins ◽

Translocator Protein ◽

Secretion Systems ◽

Yeast Two Hybrid ◽

Type Iii ◽

Yeast Two Hybrid System ◽

Two Hybrid

Many Gram-negative pathogens employ a specific secretion pathway, termed type III secretion, to deliver virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. Subsequent functions of many effector proteins delivered in this manner result in subversion of host-signalling pathways to facilitate bacterial entry, survival and dissemination to neighbouring cells and tissues. Whereas the secreted components of type III secretion systems (TTSSs) from different pathogens are structurally and functionally diverse, the structural components and the secretion apparatus itself are largely conserved. TTSSs are large macromolecular assemblies built through interactions between protein components of hundreds of individual subunits. The goal of this project was to screen, using the standard yeast two-hybrid system, pair-wise interactions between components of the enteropathogenic Escherichia coli TTSS. To this end 37 of the 41 genes encoded by the LEE pathogenicity island were cloned into both yeast two-hybrid system vectors and all possible permutations of interacting protein pairs were screened for. This paper reports the identification of 22 novel interactions, including interactions between inner-membrane structural TTSS proteins; between the type III secreted translocator protein EspD and structural TTSS proteins; between established and putative chaperones and their cognate secreted proteins; and between proteins of undefined function.

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