Area 2: Protein Interaction Technologies

(i) Mammalian Membrane Two-Hybrid Assay

Although MYTH is a robust yeast-based technique to identify protein partners of integral membrane proteins, certain proteins of mammalian origin have proven difficult to study using the technology. This is due largely to differences between yeast and mammalian protein modification systems, protein cofactors, and membrane composition.

Although several mammalian versions of the original yeast two-hybrid system have been developed, these techniques can be technically challenging and are often not suitable for high-throughput applications, especially when studying integral membrane proteins. Hence, we aim to develop a new mammalian membrane two-hybrid technology (mammMTH) that can detect protein-protein interactions in mammalian cells (Figure 1).

Like classical MYTH, the technology takes advantage of the previously described split-ubiquitin approach, though with a variety of crucial modifications. Initial proof of principle experiments have proven successful, and we are currently in the process of adapting mammMTH for use in a high-throughput format.
In summary, the mammMTH system will provide an invaluable tool for uncovering the biological role of many mammalian integral membrane proteins that cannot be studied using the classical MYTH approach. Given that many disease-related proteins are integral membrane proteins, this method has remarkable potential to advance our identification and understanding of novel pharmacological targets.

(ii) Cross-and-Capture Assay

We developed a novel assay, called Cross-and-Capture, that allows a rapid analysis of protein-protein interactions by using differentially tagged yeast arrays in the two haploid yeast mating types. In MATa cells, “ bait” ORFs are tagged at the 3' end with a sequence encoding 6 histidines (6xHIS), while “prey” ORFs in MATα cells are tagged with a sequence encoding a triple VSV tag (3xVSV). Both tags also contain a V5 epitope to allow identification of both bait and prey proteins. To examine a particular PPI, a bait strain is crossed to a prey strain to generate a diploid expressing the desired bait and prey tagged proteins.

Following diploid growth and cell lysis, extracts are incubated with nickel beads, which allows isolation of the 6xHIS tagged bait protein and proteins associated with the bait. Bound proteins are examined by immunoblot analysis for the presence of the bait and prey proteins using anti-V5 and anti-VSV antibodies. If the prey protein binds to the nickel beads in a bait-dependent manner, a PPI can be inferred between the bait and prey proteins. The combination of 6xHIS and 3xVSV tags was selected over other epitope-tags such as HA-, myc-, and FLAG-tags based on careful comparison of detection efficiency, reliability, and cost.

We applied the Cross-and-Capture system to a subset of 506 yeast ORFs. 258 of these ORFs encode proteins involved in DNA repair, replication and recombination (SGD, www.yeastgenome.org ) and another 248 ORFs encode proteins of unknown function that were assigned to the nucleus based on their localization patterns. We demonstrated that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we used “Cross-and-Capture” to identify two novel protein complexes, Rtt101p/Mms1p and Sae2p/Mre11p. Finally, in a targeted survey for posttranslational protein modifications, we showed that Pms1p, a conserved yeast protein involved in mismatch repair, is phosphorylated in the presence of DNA damage. Our studies establish the “Cross-and-Capture” assay as a novel, versatile tool for yeast proteomics.

Selected Publications

Petschnigg J., Moe O., and Stagljar I. (2011) Using yeast as a model to study membrane proteins, Curr Opin Nephrol Hypertens 20, 425-432

Suter, B., Kittanakom, S., and Stagljar, I. (2008) Two-hybrid technologies in proteomics research, Curr Opin Biotechnol 9, 316-323.

Suter, B., Graham, C.I., and Stagljar, I. (2008) Exploring protein phosphorylation in response to DNA damage using differentially tagged yeast arrays, BioTechniques 45, 581-584.

Suter, B., Fetchko, M.J. Imhof, R., Graham, C., Stoffel-Studer, I., Zbinden, C., Raghavan, M., Benetti, L., Hort, J., Filingham, J., Greenblatt, J.F., Guri N. Giaever, G.N., Nislow, C., and Stagljar, I. (2007) Examining protein-protein interactions using endogenously tagged yeast arrays: the Cross-and-Capture system, Genome Res 17, 1774-1782.

Suter, B., Auerbach, D., and Stagljar, I. (2006) Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond. BioTechniques 40 , 625-644.

Thaminy, S., Auerbach, D., Arnoldo, A., and Stagljar, I. (2003) Identification of novel ErbB3-interacting proteins using the split-ubiquitin membrane yeast two-hybrid technology, Genome Res 13, 1744-1753.

Stagljar, I., Korostensky, C., Johnsson, N. and te Heesen, S. (1998) A new genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo, Proc Natl Acad Sci USA 95, 5187-5192.