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| Title |
Rapid Mapping of Interactions between Human SNX-BAR Proteins Measured In Vitro by AlphaScreen and Single-molecule Spectroscopy*
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|---|---|
| Published in |
Molecular and Cellular Proteomics, May 2014
|
| DOI | 10.1074/mcp.m113.037275 |
| Pubmed ID | |
| Authors |
Emma Sierecki, Loes M Stevers, Nichole Giles, Mark E Polinkovsky, Mehdi Moustaqil, Sergey Mureev, Wayne A Johnston, Mareike Dahmer-Heath, Dubravka Skalamera, Thomas J Gonda, Brian Gabrielli, Brett M Collins, Kirill Alexandrov, Yann Gambin |
| Abstract |
Protein dimerisation and oligomerisation is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to information transfer in signalling, transcriptional and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allows rapid mapping of homo and hetero oligomerisation of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerisation of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pairwise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerisation partners for each family member. We find that while nine SNX-BAR proteins are able to form homodimers, all members including the retromer-associated SNX1, SNX2 and SNX5 can form selective heterodimers, while SNX2, SNX4, SNX6 and SNX8 are promiscuous binders of other SNX-BAR proteins. Remarkably, we also observed that the BAR domain lacking SNX3 interacts with SNX8 indicating a different interaction mode. We conclude that the presented combination of methods enables rapid reconstitution and analysis of protein-protein interaction networks in vitro. |
Mendeley readers
The data shown below were compiled from readership statistics for 51 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| India | 1 | 2% |
| Australia | 1 | 2% |
| Unknown | 49 | 96% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 13 | 25% |
| Student > Ph. D. Student | 10 | 20% |
| Student > Master | 7 | 14% |
| Student > Bachelor | 3 | 6% |
| Professor | 3 | 6% |
| Other | 4 | 8% |
| Unknown | 11 | 22% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 20 | 39% |
| Biochemistry, Genetics and Molecular Biology | 14 | 27% |
| Chemical Engineering | 1 | 2% |
| Unspecified | 1 | 2% |
| Medicine and Dentistry | 1 | 2% |
| Other | 1 | 2% |
| Unknown | 13 | 25% |