Rapid Mapping of Interactions between Human SNX-BAR Proteins Measured In Vitro by AlphaScreen and Single-molecule Spectroscopy

Abstract

Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, 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 allow rapid mapping of homo-and hetero-oligomerization 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. Dimerization 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 dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure. Molecular & Cellular Proteomics

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

“…Figure 4F,G show that WT α-synuclein is only able to interact with itself, G51D and A30P but not with E46K, H50Q or A53T. To verify these results and widen the test to interactions between all protein pairs, a proximity assay was used39 (Amplified Luminescent Proximity Homogeneous Assay Screen, or AlphaScreen). The AlphaScreen assay utilizes two nanobeads to detect protein-protein interactions with high sensitivity (See Fig.…”

“…The protein concentrations are calculated from the sGFP fluorescence. A calibration curve was obtained for E. Coli purified fluorescent proteins (sGFP and GFP-BBS9)39. Visualization under a confocal microscope showed homogeneous fluorescence for WT α-synuclein, but the presence of bright, small fluorescent specks diffusing in solution.…”

Formation of Tubulovesicular Carriers from Endosomes and Their Fusion to the trans-Golgi Network

“…The yeast Vps5-Vps17 SNX-BAR heterodimer is also conserved in mammals as one Vps5 ortholog dimerizes with one Vps17 homolog (Koumandou et al, 2011;Wassmer et al, 2007). Interestingly, a recent analysis of homo-and hetero-oligomerization of the human SNX-BAR protein family using a cell-free protein coexpression coupled to a fluorescence proximity assay showed that while several SNX-BAR proteins are able to form homodimers, the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization (Sierecki et al, 2014). Notably, the formation of heterodimers in the SNX-BAR-retromer complex parallels the capacity of remodeling liposomes into tubules in vitro (van Weering et al, 2012).…”

“…Here, it is important to consider that, although some SNX-BAR proteins have a propensity to participate in promiscuous interactions that could indicate nonspecific binding, the recent pairwise map of retromer-interacting SNX-BAR proteins with other members of the SNX-BAR family (Sierecki et al, 2014) hints at the possibility that the number of SNX-BAR combinations potentially associated with retromer could be significantly larger than initially anticipated. This possibility merits further exploration to understand the complexity of the mammalian retromer.…”

Biogenesis of endosome-derived transport carriers

“…These observations suggest that the vacuolar domain of the endosome [6] provides a reservoir of SNX-BAR protomers that are recruited to the growing carrier tubule by oligomerization [7], thereby promoting the growth of the budding tubule. Cataloging of human SNX-BARs on the basis of oligomerization, in vitro tubulation activity, and localization within cells suggests the potential for at least nine unique SNX-BAR coats to exist in cells (see [8, 9]). In principle, each potential SNX-BAR oligomer might confer a cargo-specific trafficking pathway, but the extent to which this is the case is yet to be determined.…”