Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions

Fetchko, Michael; Štagljar, Igor

doi:10.1016/j.ymeth.2003.10.010

Cited by 83 publications

(70 citation statements)

References 24 publications

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“…A detailed methods paper for using the membrane-bound split-ubiquitin yeast two-hybrid (MbYTH) system has been published previously (Fetchko and Stagljar, 2004). To create the UNC-84 bait construct, the sequence encoding the C-terminal 633 residues of UNC-84 was amplified using PfuUltra high-fidelity DNA polymerase (Stratagene) from the cDNA yk402g1 (a gift from Y. Kohara, National Institute of Genetics, Mishima, Japan) with overhanging NcoI and NheI restriction sites.…”

Section: Split-ubiquitin Yeast Two-hybrid Systemmentioning

confidence: 99%

“…For the N969A and P973A mutations, the sequence encoding the C-terminal KASH domain was amplified with PfuUltra using primers that incorporated the point mutations, and cloned into pNubG-HA-prey as described above. (Fetchko and Stagljar, 2004). Controls were done to ensure that neither the bait nor prey constructs self-activated the reporters and that the bait was expressed in the proper orientation ( Figure 5; Fetchko and Stagljar, 2004).…”

Section: Split-ubiquitin Yeast Two-hybrid Systemmentioning

confidence: 99%

“…The product was cloned into pNubG-HA-prey (a gift from I. Stagljar, University of Zurich-Irchel, Zurich, Switzerland) to create pSL37, which expresses aNubG::UNC-83 fusion protein and the TRP1 marker. The pNubI vector was used as a control to confirm proper orientation of the bait constructs in the membrane (gift from I. Stagljar) (Fetchko and Stagljar, 2004). The UNC-84 point mutations were made using pSL36 as a template and PCR SOEing mutagenesis (Horton et al, 1990).…”

mentioning

confidence: 99%

“…The HIS3 reporter construct was assayed by growth on SD Ϫtrp Ϫleu Ϫhis dropout plates supplemented with 2 mM 3-amino-1,2,4-triazole (Sigma-Aldrich) to prevent leakiness of the his3-200 gene. Qualitative assessment of the lacZ reporter was done by filter lift ␤-galactosidase assays (Fetchko and Stagljar, 2004). …”

mentioning

confidence: 99%

“…We hypothesized that to detect a physical interaction between the KASH domain of UNC-83 and UNC-84, the KASH domain would need to be presented in the proper context of a membrane. Thus, we used the splitubiquitin membrane-bound yeast two-hybrid (MbYTH) system, which allowed us to test for such an interaction in a more natural environment, associated with the lumenal face of the yeast endoplasmic reticulum (ER) membrane.The MbYTH system is used to study interactions between membrane proteins and uses complementation between two separable domains of the small protein ubiquitin (Figure 5;Fetchko and Stagljar, 2004). The wild-type N-terminal half of ubiquitin (NubI; amino acids 1-34) is able to spontaneously interact with the C-terminal half of ubiquitin (Cub; amino acids 35-76).…”

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confidence: 99%

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UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

McGee

Rillo

Anderson

et al. 2006

MBoC

128

180

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UNC-84 is required to localize UNC-83 to the nuclear envelope where it functions during nuclear migration. A KASH domain in UNC-83 was identified. KASH domains are conserved in the nuclear envelope proteins Syne/nesprins, Klarsicht, MSP-300, and ANC-1. Caenorhabditis elegans UNC-83 was shown to localize to the outer nuclear membrane and UNC-84 to the inner nuclear membrane in transfected mammalian cells, suggesting the KASH and SUN protein targeting mechanisms are conserved. Deletion of the KASH domain of UNC-83 blocked nuclear migration and localization to the C. elegans nuclear envelope. Some point mutations in the UNC-83 KASH domain disrupted nuclear migration, even if they localized normally. At least two separable portions of the C-terminal half of UNC-84 were found to interact with the UNC-83 KASH domain in a membrane-bound, split-ubiquitin yeast two-hybrid system. However, the SUN domain was essential for UNC-84 function and UNC-83 localization in vivo. These data support the model that KASH and SUN proteins bridge the nuclear envelope, connecting the nuclear lamina to cytoskeletal components. This mechanism seems conserved across eukaryotes and is the first proposed mechanism to target proteins specifically to the outer nuclear membrane. INTRODUCTIONA variety of cellular and developmental processes, including fertilization, cell division, cell migration, and establishment of polarity, depend on positioning the nucleus to a specific location within the cell. For example, in budding yeast the nucleus must migrate to the bud neck before the onset of mitosis. Also, nuclei actively follow the leading edge of migratory cells, such as those in the developing cerebral cortex. Nuclear migration defects in these two examples lead to the missegregation of chromosomes or the neurological disease lissencephaly, respectively (reviewed in Morris, 2000). The role of microtubules and associated dynein and kinesin motors in nuclear migration are well established (reviewed in Reinsch and Gonczy, 1998). Although less established, actin also plays an important role in many nuclear positioning events (reviewed in Starr and Han, 2003). Recently, a definitive role for actin networks has been described in nuclear migration during NIH 3T3 cell polarization (Gomes et al., 2005). It remains relatively unknown how the nucleus connects to the cytoplasmic cytoskeleton during nuclear migration. Furthermore, it is not clear how the forces involved in nuclear positioning are transferred across both membranes of the nuclear envelope from the cytoskeleton to the nuclear matrix.We have previously proposed that two C. elegans proteins, UNC-84 and UNC-83, function to control nuclear migration by bridging the nuclear envelope, connecting the cytoskeleton with the nuclear matrix (Starr et al., 2001;Lee et al., 2002;Starr and Han, 2003). Mutations in unc-83 or unc-84 disrupt nuclear migration in at least three cell types: embryonic hypodermal hyp7 precursors, larval hypodermal P-cells, and embryonic intestinal primordial cells (Horvitz and Sulst...

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Section: Split-ubiquitin Yeast Two-hybrid Systemmentioning

confidence: 99%

Section: Split-ubiquitin Yeast Two-hybrid Systemmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

McGee

Rillo

Anderson

et al. 2006

MBoC

128

180

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Methods of Analysis for Imaging and Detecting Ions and Molecules

Kim

Tao

Umezawa

2009

Cellular and Biomolecular Recognition

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A Simple Quantitative Assay for Measuring β‐Galactosidase Activity Using X‐Gal in Yeast‐Based Interaction Analyses

2022

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Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme β-galactosidase (β-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) is an inexpensive and sensitive substrate of β-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5 -dibromo-4,4 -dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (orthonitrophenyl-β-galactopyranoside) as a substrate for β-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression.

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Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions

Cited by 83 publications

References 24 publications

UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

UNC-83 Is a KASH Protein Required for Nuclear Migration and Is Recruited to the Outer Nuclear Membrane by a Physical Interaction with the SUN Protein UNC-84

Methods of Analysis for Imaging and Detecting Ions and Molecules

A Simple Quantitative Assay for Measuring β‐Galactosidase Activity Using X‐Gal in Yeast‐Based Interaction Analyses

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