scholarly journals Targeting signals and subunit interactions in coated vesicle adaptor complexes.

1995 ◽  
Vol 131 (3) ◽  
pp. 619-630 ◽  
Author(s):  
L J Page ◽  
M S Robinson
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Coated Vesicle ◽  
Yeast Two Hybrid ◽  
Subunit Interactions ◽  
Yeast Two Hybrid System ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Two Hybrid ◽  
Terminal Domains

There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.

scholarly journals Subunit Interactions in the mariner Transposase

Genetics ◽  
1996 ◽  
Vol 144 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
Allan R Lohe ◽  
David T Sullivan ◽  
Daniel L Hartl
Keyword(s):  
Hybrid System ◽  
Catalytic Domain ◽  
Wild Type ◽  
Target Element ◽  
Yeast Two Hybrid ◽  
Subunit Interactions ◽  
Hsp70 Promoter ◽  
Yeast Two Hybrid System ◽  
Transposition Reaction ◽  
Two Hybrid

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.

scholarly journals The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg–HCPro and VPg–VPg interactions

2003 ◽  
Vol 84 (10) ◽  
pp. 2861-2869 ◽  
Author(s):  
Ma. Leonora M. Yambao ◽  
Chikara Masuta ◽  
Kenji Nakahara ◽  
Ichiro Uyeda
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Yellow Vein ◽  
Transcription Activator ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Clover Yellow Vein Virus ◽  
Two Hybrid ◽  
Far Western Blot

Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro–VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro–VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro–VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg–VPg interaction and the central 19 amino acids were needed for the HCPro–VPg interaction.

scholarly journals Effect of mutations within the Cys-rich region of potyvirus helper component-proteinase on self-interaction

1999 ◽  
Vol 80 (11) ◽  
pp. 2809-2812 ◽  
Author(s):  
Silvio Urcuqui-Inchima ◽  
Ivan G. Maia ◽  
Gabrièle Drugeon ◽  
Anne-Lise Haenni ◽  
Françoise Bernardi
Keyword(s):  
Amino Acids ◽  
Potato Virus Y ◽  
Yeast Two Hybrid ◽  
Rich Region ◽  
Conserved Residues ◽  
Helper Component ◽  
Yeast Two Hybrid System ◽  
Terminal Amino ◽  
Helper Component Proteinase ◽  
Two Hybrid

The first ∼60 amino acids of the N-terminal part of the potyvirus helper component-proteinase (HC-Pro) include highly conserved residues comprising a Cys-rich region. In the present study, the domain in Potato virus Y sufficient for self-interaction was mapped using the yeast two-hybrid system to the 83 N-terminal amino acids of HC-Pro. Mutations in the conserved His and two Cys residues within the Cys-rich region have a strong debilitating effect on self-interaction when introduced in the full-length HC-Pro, but not when introduced in the N-terminal fragment.

scholarly journals Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3

1999 ◽  
Vol 73 (7) ◽  
pp. 5411-5421 ◽  
Author(s):  
Maria Piron ◽  
Thierry Delaunay ◽  
Jeanne Grosclaude ◽  
Didier Poncet
Keyword(s):  
Amino Acids ◽  
Hybrid System ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Domain ◽  
Initiation Factor ◽  
Yeast Two Hybrid ◽  
Dimerization Domain ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3′ end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.

scholarly journals γ-Synergin

1999 ◽  
Vol 146 (5) ◽  
pp. 993-1004 ◽  
Author(s):  
Lesley J. Page ◽  
Penelope J. Sowerby ◽  
Winnie W.Y. Lui ◽  
Margaret S. Robinson
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Yeast Two Hybrid ◽  
Eh Domain ◽  
Library Screen ◽  
Alternatively Spliced ◽  
Vesicle Cycle ◽  
Two Hybrid

The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the γ-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both γ-adaptin and α-adaptin, and γ-synergin, an alternatively spliced protein with an apparent molecular mass of ∼110–190 kD, which only interacts with γ-adaptin. γ-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of γ-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the γ-adaptin binding site. In cells expressing α-adaptin with the γ-adaptin ear, a construct that goes mainly to the plasma membrane, much of the γ-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that γ-synergin links the AP-1 complex to another protein or proteins.

scholarly journals P1 ParB Domain Structure Includes Two Independent Multimerization Domains

1999 ◽  
Vol 181 (19) ◽  
pp. 5898-5908 ◽  
Author(s):  
Jennifer A. Surtees ◽  
Barbara E. Funnell
Keyword(s):  
Amino Acids ◽  
Domain Structure ◽  
Cross Linking ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Self Association ◽  
Two Hybrid ◽  
Chemical Cross Linking

ABSTRACT ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.

scholarly journals Study of the N-Terminal Domain Homodimerization in Human Proteins with Zinc Finger Clusters

2021 ◽  
Vol 499 (1) ◽  
pp. 257-259
Author(s):  
D. V. Fursenko ◽  
P. G. Georgiev ◽  
A. N. Bonchuk
Keyword(s):  
Zinc Finger ◽  
Large Family ◽  
Yeast Two Hybrid ◽  
Long Distance ◽  
Yeast Two Hybrid System ◽  
N Terminus ◽  
Human Proteins ◽  
Two Hybrid ◽  
Architectural Protein ◽  
Terminal Domains

Abstract CTCF belongs to a large family of transcription factors with clusters of C2H2-type zinc finger domains (C2H2 proteins) and is a main architectural protein in mammals. Human CTCF has a homodimerizing unstructured domain at the N-terminus which is involved in long-distance interactions. To test the presence of similar N-terminal domains in other human C2H2 proteins, a yeast two-hybrid system was used. In total, the ability of unstructured N-terminal domains to homodimerize was investigated for six human C2H2 proteins with an expression profile similar to CTCF. The data indicate the lack of the homodimerization ability of these domains. On the other hand, three C2H2 proteins containing the structured domain DUF3669 at the N-terminus demonstrated homo- and heterodimerization activity.

scholarly journals Subunit Interactions in the Na,K-ATPase Explored with the Yeast Two-hybrid System

1997 ◽  
Vol 272 (19) ◽  
pp. 12366-12372 ◽  
Author(s):  
Thomas E. Colonna ◽  
Long Huynh ◽  
Douglas M. Fambrough
Keyword(s):  
Hybrid System ◽  
Yeast Two Hybrid ◽  
Subunit Interactions ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals The C-Terminal Domain of DnaQ Contains the Polymerase Binding Site

1999 ◽  
Vol 181 (9) ◽  
pp. 2963-2965 ◽  
Author(s):  
Sharon A. Taft-Benz ◽  
Roel M. Schaaper
Keyword(s):  
Amino Acids ◽  
Dna Polymerase ◽  
Dna Polymerase Iii ◽  
Yeast Two Hybrid ◽  
Α Subunit ◽  
Terminal Domain ◽  
Yeast Two Hybrid System ◽  
Polymerase Binding ◽  
Pol Iii ◽  
Two Hybrid

ABSTRACT The Escherichia coli dnaQ gene encodes the 3′→5′ exonucleolytic proofreading (ɛ) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that ɛ might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (α) subunit. We have created truncated forms ofdnaQ resulting in ɛ subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain ɛ subunits with the α and θ subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the α subunit while retaining normal binding to the θ subunit. In contrast, the NΔ186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the α subunit but was defective in binding to the θ subunit. A strain carrying the dnaQ991allele exhibited a strong, recessive mutator phenotype, as expected from a defective α binding mutant. The data are consistent with the existence of two functional domains in ɛ, with the C-terminal domain responsible for polymerase binding.

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