A novel in vivo assay for the analysis of protein-protein interaction

Maroun, Marlene; Aronheim, Ami

doi:10.1093/nar/27.13.e4-i

Cited by 7 publications

(3 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As an alternative, translocation-based methods have been described for detecting protein−protein interactions in living cells − . These methods use baits fused to proteins that, for instance, change distribution within the cell following a stimulus.…”

mentioning

confidence: 99%

Analysis of Protein Complex Hierarchy in Living Cells

Piljić

Schultz

2008

ACS Chem. Biol.

View full text Add to dashboard Cite

The identification and analysis of protein complexes is usually achieved by performing in vitro experiments. We describe a translocation-based method for studying protein complexes in living cells. Annexin A4, a phospholipid- and calcium-binding protein, translocates from the cytoplasm and nucleoplasm to plasma and nuclear membranes in response to elevated intracellular calcium levels. By fusing a bait protein, for instance, a core component of a protein complex or a similarly relevant peptide, to the annexin, translocation of both bait and its interacting target proteins are readily monitored in living cells in response to a single treatment. Proteins of interest are fused to a variety of fluorescent proteins suitable for multiparameter imaging. Using this generally applicable approach, we were able to visualize the formation of protein complexes in their natural environment. Specifically, we detected the hierarchical assembly of four-component protein complexes in single cells.

show abstract

mentioning

confidence: 99%

Analysis of Protein Complex Hierarchy in Living Cells

Piljić

Schultz

2008

ACS Chem. Biol.

View full text Add to dashboard Cite

show abstract

“…temperature revertants (102), growth at suboptimal temperatures, and the obligatory replica plating step (281), but they nevertheless remain popular and very successful alternatives to the original two-hybrid system (e.g., see references 147, 157, 256, 331, 455, 647, and 692). The Ras recruitment system was further developed for use in mammalian cells (432). Trimeric-G-protein-based methods.…”

Section: Membrane-localized and Secretory Pathway Two-hybrid Systemsmentioning

confidence: 99%

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Stynen

Tournu

Tavernier

et al. 2012

Microbiol Mol Biol Rev

181

143

View full text Add to dashboard Cite

SUMMARY The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli , but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays.

show abstract

“…However, similar systems have been developed for use in mammalian and prokaryotic cells (Mohler & Blau, 1996;Luo et al, 1997;Joung, Ramm, & Pabo, 2000;Serebriiskii, Toby, & Golemis, 2000). In addition, a Ras-recruitment system has been developed for use in mammalian cell lines (Maroun & Aronheim, 1999).…”

Section: Other Two-hybrid Systemsmentioning

confidence: 99%

Studying the interactome with the yeast two‐hybrid system and mass spectrometry

Causier

2004

Mass Spectrometry Reviews

View full text Add to dashboard Cite

I. Introduction 00 II. The Yeast Two‐Hybrid System 00 A. A Brief History 00 1. Basis of Yeast Two‐Hybrid System 00 2. Development of the System 00 B. Current Systems 00 1. The GAL4 and LexA Systems 00 2. The Sos Recruitment System 00 3. The Split‐Ubiquitin System 00 4. The Dual‐Bait System 00 5. Other Two‐Hybrid Systems 00 6. Variations on a Theme 00 C. Applications of Yeast Two‐Hybrid 00 III. Studying Protein Interactions by MS 00 IV. Genome‐Wide Analysis of Protein–Protein Interactions 00 A. Yeast Two‐Hybrid 00 B. Mass Spectrometry 00 C. Comparison of the Two Approaches 00 D. Placing Interactions in Context 00 V. Emerging Technologies 00 VI. Conclusions 00 Acknowledgments 00 Abbreviations 00 References 00 Protein interactions are crucial to the life of a cell. The analysis of such interactions is allowing biologists to determine the function of uncharacterized proteins and the genes that encode them. The yeast two‐hybrid system has become one of the most popular and powerful tools to study protein–protein interactions. With the advent of proteomics, the two‐hybrid system has found a niche in interactome mapping. However, it is clear that only by combining two‐hybrid data with that from complementary approaches such as mass spectrometry (MS) can the interactome be analyzed in full. This review introduces the yeast two‐hybrid system to those unfamiliar with the technique, and discusses how it can be used in combination with MS to unravel the network of protein interactions that occur in a cell. © 2004 Wiley Periodicals, Inc., Mass Spec Rev

show abstract

A novel in vivo assay for the analysis of protein-protein interaction

Cited by 7 publications

References 31 publications

Analysis of Protein Complex Hierarchy in Living Cells

Analysis of Protein Complex Hierarchy in Living Cells

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Studying the interactome with the yeast two‐hybrid system and mass spectrometry

Contact Info

Product

Resources

About