Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins

Golemis, Erica A.; Serebriiskii, Ilya G.; Finley, Russell L.; Kolonin, Mikhail G.; Gyuris, Jenő; Brent, Roger

doi:10.1002/0471140864.ps1902s14

Cited by 58 publications

(112 citation statements)

References 39 publications

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“…The LexA two-hybrid system (Estojak et al, 1995;Golemis et al, 1996) was used as described previously (Eugster et al, 2000). Yeast reporter strains EGY48 (Table 1), as well as the bait plasmids pEG202 and the prey plasmids pJG4-5 were used (Golemis et al, 1996).…”

Section: Fusion Constructs; Two-hybrid Assaysmentioning

confidence: 99%

“…Yeast reporter strains EGY48 (Table 1), as well as the bait plasmids pEG202 and the prey plasmids pJG4-5 were used (Golemis et al, 1996). Prey fusions were under the control of the GAL1 promoter.…”

Section: Fusion Constructs; Two-hybrid Assaysmentioning

confidence: 99%

“…Fusion constructs were created by ligating PCR products made using custom primers into vectors mentioned above, generating in frame fusions with LexA (in the bait vector) or the "acid blob B42Ј (in the prey vector)." Reporter yeast strain EGY48 (harboring URA3 plasmid pSH18-34; Golemis et al, 1996), was cotransformed with a bait and a prey fusion construct and transformants were selected on ϪHIS, ϪTRP, ϪURA plates. To assess growth on media lacking leucine, transformants were streaked onto plates lacking histidine, tryptophane, uracil, and leucine, containing 2% galactose/1% raffinose or 2% glucose, and incubated at 24 or 30°C for 3 or 4 d, respectively.…”

Section: Fusion Constructs; Two-hybrid Assaysmentioning

confidence: 99%

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The α- and β′-COP WD40 Domains Mediate Cargo-selective Interactions with Distinct Di-lysine Motifs

Eugster

Frigerio

Dale

et al. 2004

MBoC

107

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Coatomer is required for the retrieval of proteins from an early Golgi compartment back to the endoplasmic reticulum. The WD40 domain of ␣-COP is required for the recruitment of KKTN-tagged proteins into coatomer-coated vesicles. However, lack of the domain has only minor effects on growth in yeast. Here, we show that the WD40 domain of ␤ -COP is required for the recycling of the KTKLL-tagged Golgi protein Emp47p. The protein is degraded more rapidly in cells with a point mutation in the WD40 domain of ␤ -COP (sec27-95) or in cells lacking the domain altogether, whereas a point mutation in the Clathrin Heavy Chain Repeat (sec27-1) does not affect the turnover of Emp47p. Lack of the WD40 domain of ␤ -COP has only minor effects on growth of yeast cells; however, absence of both WD40 domains of ␣-and ␤ -COP is lethal. Two hybrid studies together with our analysis of the maturation of KKTN-tagged invertase and the turnover of Emp47p in ␣-and ␤ -COP mutants suggest that the two WD40 domains of ␣-and ␤ -COP bind distinct but overlapping sets of di-lysine signals and hence both contribute to recycling of proteins with di-lysine signals.

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Section: Fusion Constructs; Two-hybrid Assaysmentioning

confidence: 99%

Section: Fusion Constructs; Two-hybrid Assaysmentioning

confidence: 99%

Section: Fusion Constructs; Two-hybrid Assaysmentioning

confidence: 99%

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The α- and β′-COP WD40 Domains Mediate Cargo-selective Interactions with Distinct Di-lysine Motifs

Eugster

Frigerio

Dale

et al. 2004

MBoC

107

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“…Plasmids pRFHM1 and pSH17-4 were used as controls for the assay. The two-hybrid assay was performed essentially as described previously (Golemis et al, 1998). For each interaction, at least three individual transformants were tested.…”

Section: Two-hybrid Assaymentioning

confidence: 99%

Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

Knop

Miller

Mazza

et al. 2005

MBoC

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In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p-Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins.These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery. INTRODUCTIONEvolutionarily conserved molecular machinery regulates transport vesicle targeting, tethering, and fusion in eukaryotic cells. In yeast Saccharomyces cerevisiae this machinery involves the activity of the eight-subunit (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p) tethering complex, the exocyst, a rab family small GTPase Sec4p, and the exocytic SNARE complex (Snc1/2p, Sec9p, and Sso1/2p) thought to drive the actual lipid bilayer fusion . The exocyst subunit Sec15p has been shown to act as an effector for Sec4p (Guo et al., 1999). In addition, sec4-8 mutant cells are defective in Snc1/2p-Sec9p-Sso1/2p SNARE complex assembly (Carr et al., 1999). Despite these and numerous other studies, the regulatory mechanisms for SNARE complex formation are still poorly understood. The Sec1/Munc18 (SM) protein family members represent central regulators of SNARE complex function. These proteins perform an essential, albeit currently poorly understood function in SNARE complex regulation (Gallwitz and Jahn, 2003;Toonen and Verhage, 2003;Kauppi et al., 2004).Yeast S. cerevisiae possesses four Sec1p-family proteins: Sec1p mediates vesicle fusion at the plasma membrane (Novick et al., 1981;Carr et al., 1999), Sly1p mediates vesicle fusion at the endoplasmic reticulum-Golgi interface (Ossig et al., 1991), Vps33p is required for endosome to vacuole transport and vacuole maintenance (Banta et al., 1990), and Vps45p mediates Golgi-to-vacuole transport (Piper et al., 1994;Cowles et al., 1994). Sec1p is the closest homologue of the mammalian Munc18-1 that has been shown to associate with syntaxin 1 and to regulate the syntaxin 1-synaptobrevin-SNAP-25 SNARE complex assembly (Misura et al., 2000;Kaup...

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“…The PCR product was digested with NcoI (underlined) and BamHI (present in the TBP sequence) and cloned into pEG202-94ATT (24). LexA-TBP sequences were excised with BamHI and SphI.…”

Section: Plasmidsmentioning

confidence: 99%

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

Ryan

Stafford

et al. 2000

Molecular and Cellular Biology

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Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing the HIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at the CHA1 promoter. Finally, we show that activation of the GAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.Transcriptional activators are thought to stimulate transcription of TATA-containing promoters in part by recruiting TFIID, a multiprotein complex consisting of the TATA-binding protein (TBP) and TBP-associated factors (TAFs), to the TATA box (25,60). Several in vitro and in vivo studies support this model. For example, transcription initiated at a mutated TATA element by induced synthesis of TBP with altered specificity was enhanced in both rate and extent in the presence of an activator that could bind upstream of the relevant promoter, consistent with activator-mediated recruitment (40). Recruitment has also been inferred from the results of "activator bypass" experiments in which artificial recruitment of TBP to promoter sites near the TATA element resulted in transcriptional activation, implying that TBP recruitment is a rate-limiting step in transcriptional activation in vivo (10,39,79). Most convincingly, chromatin immunoprecipitation experiments revealed TBP to be physically associated with promoters of numerous genes under activated, but not nonactivated, conditions, implying that recruitment accompanies activation (43,46).A potential obstacle to recruitment of TBP in vivo is posed by chromatin. Binding of TBP to nucleosomal TATA elements is greatly impeded in vitro (23, 32). Transcription is also strongly repressed by chromatin, but this repression can be largely alleviated by binding of act...

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Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins

Cited by 58 publications

References 39 publications

The α- and β′-COP WD40 Domains Mediate Cargo-selective Interactions with Distinct Di-lysine Motifs

The α- and β′-COP WD40 Domains Mediate Cargo-selective Interactions with Distinct Di-lysine Motifs

Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

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