Split‐Ubiquitin and the Split‐Protein Sensors: Chessman for the Endgame

Müller, Judith; Johnsson, Nils

doi:10.1002/cbic.200800190

Cited by 56 publications

(44 citation statements)

References 74 publications

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“…The split-ubiquitin system is the pioneering, classic example of a PCA method (Johnsson and Varshavsky, 1994;Müller and Johnsson, 2008), a categorization that is important for obtaining an in-depth understanding of the methods, their underlying biology, and potential limitations. While the two-hybrid system in principle can be converted to a yeast one-hybrid system, allowing the detection of protein-DNA interactions (Gstaiger et al, 1995), the PCA approach requires both split reporter fragments for a meaningful output; each fragment by itself is non-functional, an important difference.…”

Section: Categories Of Ppi Techniquesmentioning

confidence: 99%

“…c It is estimated that the NubI13A mutation increases the K d to 1 mM and that the NubI13G mutation value might be even higher, allowing the split-ubiquitin system to detect transient interactions up to K d values of 100 mM (Müller and Johnsson, 2008). The split fluorescent protein fragments are known to spontaneously and irreversibly reassociate; hence, the K d is negligibly small (Magliery et al, 2005).…”

Section: Box IImentioning

confidence: 99%

“…4D; Wittke et al, 1999). Rapid turnover of URA3 renders cells auxotrophic to uracil but resistant to 5-fluoroorotic acid (Müller and Johnsson, 2008). Hence, the URA3-based splitubiquitin system can be used for either the detection of binary PPIs (when counterselection with 5-fluoroorotic acid is used) or the screening of inhibitors or point mutations that abolish an interaction (when using uracildepleted medium for selection).…”

Section: The Split-ubiquitin System: a Full-length Alternativementioning

confidence: 99%

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Techniques for the analysis of protein-protein interactions in vivo

et al. 2016

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ORCID ID: 0000-0002-5820-4466 (C.G.).Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of crossspecies networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique.

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Section: Categories Of Ppi Techniquesmentioning

confidence: 99%

Section: Box IImentioning

confidence: 99%

Section: The Split-ubiquitin System: a Full-length Alternativementioning

confidence: 99%

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Techniques for the analysis of protein-protein interactions in vivo

et al. 2016

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“…Most existing FPs have been used to create split reporters and have been used to investigate the dynamics of many protein-protein interactions (reviewed in Muller and Johnsson, 2008). Upon protein interaction, the two FP fragments come into close proximity and the FP is reconstituted.…”

Section: Protein-protein Interactionsmentioning

confidence: 99%

From jellyfish to biosensors: the use of fluorescent proteins in plants

Voß¹,

Larrieu²,

Wells³

2013

Int. J. Dev. Biol.

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The milestone discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria, its optimisation for efficient use in plantae, and subsequent improvements in techniques for fluorescent detection and quantification have changed plant molecular biology research dramatically. Using fluorescent protein tags allows the temporal and spatial monitoring of dynamic expression patterns at tissue, cellular and subcellular scales. Genetically-encoded fluorescence has become the basis for applications such as cell-type specific transcriptomics, monitoring cell fate and identity during development of individual organs or embryos, and visualising protein-protein interactions in vivo. In this article, we will give an overview of currently available fluorescent proteins, their applications in plant research, the techniques used to analyse them and, using the recent development of an auxin sensor as an example, discuss the design principles and prospects for the next generation of fluorescent plant biosensors.

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“…In addition, it has now been shown that gcr2 mutants, as well as mutants in two related genes, gcr2-like1 and gcr2-like2, and the various double and triple mutant combinations all failed to show the reported ABA response in stomatal regulation, seed germination, or early seedling development, unlike that seen in loss-of-function gpa1 mutants Guo et al, 2008). These results raise questions about the validity of the methods used to demonstrate an interaction with GPA1; in particular, others have emphasized the need for care when assessing the interaction of membrane proteins using ''split-protein sensors'' (Mü ller and Johnsson, 2008). Our results, together with these reports that contradict the findings of Liu et al (2007), demonstrate that GCR2 is not an extracellular ABA receptor.…”

mentioning

confidence: 99%