Part:BBa_K5466008

NubG

N-terminal part of the split ubiquitin (residues 1-34) with a flexible linker. It can re-associate with Cub (BBa_K5466009) in vivo and form active ubiquitin. Use in the assessment of membrane protein intaraction, through the split ubiquitin system, a membrane based Yeast-Two Hybrid.

The intracellular and signaling region of a receptor inspired in GEMS platform: Patrol Yeast.

Spontaneous reconstitution of the split protein is prevented through mutation I13G, rendering NubG. Only when NubG and Cub are brought into close proximity upon receptor dimerization, can the ubiquitin be reconstituted and recognized by cellular deubiquitinases, leading to hydrolysis and release of a transcription factor fused to the C-terminal end of Cub.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Yeast-two hybrid

This technique is designed to detect whether two proteins, referred to as "prey" and "bait", interact with each other, thanks to their fusion to the DNA-binding (DB) or activation domain (AD) of a split transcription factor. For instance, integral membrane proteins that reside within the lipid bilayer are excluded because they are unlikely to enter the nucleus, and if they do, they may become misfolded. That leads us to the need for a membrane based Yeast-Two Hybrid.

Split-ubiquitin system

Similar to the two-hybrid system, which involves the reassembly of a transcription factor upon the interaction of two test proteins, the split-ubiquitin system is composed of two fragments of ubiquitin that come together through interacting proteins. Ubiquitin is a small protein of 76 amino acids and plays a crucial role in various cellular pathways related to protein degradation. Specifically, the C-terminus of ubiquitin forms an amide bond with proteins designated for degradation, facilitated by a complex system of several enzymes. Ubiquitin-specific proteases identify the ubiquitin portion of these protein fusions and cleave the covalent bond linking ubiquitin to the protein. The resulting protein is then targeted for degradation by the proteasome.

In the split-ubiquitin system for detecting interactions, ubiquitin is expressed as two distinct fragments in separate plasmids: the N-terminal fragment, NubI, and the C-terminal fragment, Cub. A reporter protein is linked to the C-terminus of Cub, and when NubI is co-expressed, the two fragments of ubiquitin reassemble, forming a split-ubiquitin heterodimer that leads to the release of the reporter protein. This occurs because split-ubiquitin acts as a substrate for ubiquitin-specific proteases, which cleave the reporter protein from the C-terminus of Cub, enabling it to enter the nucleus and activate reporter genes. In this system, the N-terminal fragment NubI has been modified with a point mutation, which does not spontaneously associate with Cub. Instead, NubG and the Cub-reporter can only reassemble when the fusion proteins attached to them interact.

Since the formation of the split-ubiquitin complex does not necessitate specific localization of the proteins, the system has been adapted to investigate interactions between membrane proteins. In the membrane-based yeast two-hybrid system, one protein of interest, known as bait X, is fused to the Cub domain, followed by an artificial transcription factor (TF). The other protein, referred to as prey Y, is fused to NubG. When both proteins interact, they promote the assembly of the split-ubiquitin complex and trigger the proteolytic release of the TF, allowing it to enter the nucleus and activate reporter genes.

Fuse NubG with the "prey" protein for interaction study. To complete the split ubiquitin system you need to fuse Cub (BBa_K5466009) with the "bait" protein.

Yeast Patrol

This techniques have been repurposed for application as synthetic receptors by substituting the “bait” and “prey” proteins with scFvs or sdAbs , which can promote ligand-induced dimerization of fused domains, restoring their ability to activate gene expression and producing a response to the target.

One of this approaches, named Patrol Yeast, drew inspiration from GEMS on the use of EpoR as a receptor scaffold to create a signaling platform in S. cerevisiae based on the split-ubiqutin system. This resulted in a system composed of LBD-EpoR-NubG and LBD-EpoR-Cub-TF proteins which was demonstrated to admit scFvs and sdAbs (including VL and VH chains) as ligand binding domains

To construct a Yeast Patrol receptor:

Fuse NubG with EpoR (BBa_K5466007) and ligand binding domain (LBD) to construct a receptor.

To complete the signaling platform you need to fuse Cub (BBa_K5466009), EpoR (BBa_K5466007) and a LBD distinct from the one used in the NubG, but that specifically binds to the ligand (unless the same LBD can dimerize in the presence of the ligand).

You can fuse it with a golden gate assembly without scars and in frame.

References

Ivanusic, D., Heinisch, J. J., Eschricht, M., Laube, U., & Denner, J. (2015). Improved split-ubiquitin screening technique to identify surface membrane protein-protein interactions. BioTechniques, 59(2), 63-73. https://doi.org/10.2144/000114315

Su, J., Zhu, B., Inoue, A., Oyama, H., Morita, I., Dong, J., Yasuda, T., Sugita-Konishi, Y., Kitaguchi, T., Kobayashi, N., Miyake, S., & Ueda, H. (2022). The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning. Biosensors And Bioelectronics, 219, 114793. https://doi.org/10.1016/j.bios.2022.114793

Thaminy, S., Miller, J., & Stagljar, I. (2004). The Split-Ubiquitin Membrane-Based Yeast Two-Hybrid system. Humana Press eBooks, 297–312. https://doi.org/10.1385/1-59259-762-9:297