If you’ve read our article, An Overview of Yeast Two-Hybrid (Y2H) Screening, you’ll know that one major limitation of conventional Y2H is that your protein-protein interaction must occur in the nucleus for the reporter gene to be activated.

So what do you do if your protein is a receptor tyrosine kinase? Or a G protein–coupled receptor, membrane-bound phosphatase, or cell-surface transporter? All of these proteins represent important classes of signalling molecules – but they aren’t nuclear, and so can’t be studied using conventional Y2H.

In this article we’ll cover one Y2H modification for the detection of protein-protein interactions between non-nuclear or insoluble proteins – the split ubiquitin yeast two-hybrid assay.

Like a conventional Y2H, the split ubiquitin Y2H relies on separating the functional regions of a protein into modular domains – except instead of splitting a transcription factor, it uses a split ubiquitin protein.

What is ubiquitin?

Ubiquitin is a small, highly conserved protein that participates in the cellular signalling pathways leading to the degradation of proteins. The ubiquitin protein covalently attaches to a target protein and marks that protein for degradation by the 26S proteasome. Both the N-terminus and the C-terminus of the ubiquitin molecule are required to correctly perform this function. However, if the ubiquitin protein is split into two halves, close physical association of these two polypeptide halves is sufficient to restore their function.

How exactly does the split-ubiquitin Y2H work?

The ubiquitin protein is split into two halves – the N-terminal domain (Nub, from N-ubiquitin, amino acids 1-34), and the C-terminal domain (Cub, from C-ubiquitin, amino acids 35-76). These halves are each fused to a protein – one to your bait, and one to your prey – to form hybrid-proteins products. The Cub peptide is also bound to a reporter transcription factor. When the Cub and Nub halves are brought together into close proximity, they associate to form the full-length ubiquitin and the reporter transcription factor is released by ubiquitin specific proteases. The free transcription factor then enters the nucleus and activates transcription of reporter genes.

Decided you want to give the split ubiquitin Y2H assay a try? Here’s what you’ll need:

  1. The Nub and Cub sequences. Nub comes in two different flavors: NubI (wild-type sequence, with isoleucine at amino acid position 13), and NubG (which instead has a glycine at amino acid position 13). You’ll need both – NubI as a positive-control, and NubG for your test assays. This is because the wild-type NubI spontaneously associates with Cub when they are both expressed, but NubG has lower binding affinity for Cub and will therefore only interact when there are additional contacts from the interaction of your bound interactor proteins.
  2. A bait protein and one or more prey proteins, all of which are normally localized to the cytosol or plasma membrane. Upon interaction of the bait and prey proteins, the two halves of ubiquitin are brought together, allowing the formation of a full-length, functional ubiquitin protein. In this conformation, the ubiquitin protein is recognized by ubiquitin specific proteases that cleave off the reporter transcription factor. Speaking of which, you’ll also need…
  3. The reporter transcription factor fused to the hybrid Cub and membrane protein of interest. These transcription factors are designed to specifically activate reporter genes that have been stably integrated into the host yeast genome. Often this transcription factor is not naturally occurring; for example, a hybrid transcription factor made up of the bacterial DNA-binding protein LexA, followed by the transcription activation domain of Herpes simplex VP16, which can activate the transcription of the HIS3 and LacZ reporter genes.
  4. All the other reagents and equipment you would need for a standard Y2H, such as genetically modified yeast, selection markers, and yeast growth media. Check out our Overview of Yeast Two-Hybrid (Y2H) Screening for more details and some help on how to design your experiment.

So why choose a split ubiquitin Y2H?

Because the principles and techniques are largely the same as a conventional Y2H, the split ubiquitin Y2H shares some of the disadvantages; namely, high background, defects in post-translational modifications due to the non-mammalian expression system, and generation of purely qualitative data. However, the split ubiquitin Y2H offers the huge advantage of being able to measure interactions between insoluble, hydrophobic proteins (such as integral membrane proteins) and non-nuclear proteins. If your protein of interest falls into those categories, then this assay is for you!

Have you used a split ubiquitin Y2H assay? How did it help your research?

Useful papers

Stagljar, I., Fields, S., 2002. Analysis of membrane protein interactions using yeast-based technologies. Trends Biochem. Sci. 27, 559–563.
Stynen, B., Tournu, H., Tavernier, J., Dijck, P.V., 2012. Diversity in Genetic In Vivo Methods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System. Microbiol. Mol. Biol. Rev. 76, 331–382. doi:10.1128/MMBR.05021-11

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