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. 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. The last decade has witnessed an unprecedented increase in the number of identified human protein–protein interactions (PPIs) 1The abbreviations used are: PPI, protein–protein interaction; AlphaScreen, Amplified Luminescent Proximity Homogeneous Assay Screen; BAR, Bin/Amphiphysin/Rvs; C, coincidence; cps, count per second; G, intensity of GFP fluorescence; GFP, green fluorescent protein; GW, Gateway technology; IP (co-), immunoprecipitation (co-); LTE, Leishmania tarentolae extract; PX, Phox; SH3, SRC homology 3; SNX, sorting nexin; R, intensity of Cherry fluorescence; RT, room temperature. 1The abbreviations used are: PPI, protein–protein interaction; AlphaScreen, Amplified Luminescent Proximity Homogeneous Assay Screen; BAR, Bin/Amphiphysin/Rvs; C, coincidence; cps, count per second; G, intensity of GFP fluorescence; GFP, green fluorescent protein; GW, Gateway technology; IP (co-), immunoprecipitation (co-); LTE, Leishmania tarentolae extract; PX, Phox; SH3, SRC homology 3; SNX, sorting nexin; R, intensity of Cherry fluorescence; RT, room temperature., and these commonly form sophisticated interaction networks that mediate adaptive signaling responses to environmental stimuli. Validation of PPIs in general remains challenging, particularly when examining interactions within a family of proteins. Self-interaction is even more difficult to detect but can play an important physiological role. In human cells homo- or hetero-dimerization is a common feature of proteins regulating cell signaling, including tyrosine kinase receptors, G-protein coupled receptors, chemokines, cytokines, and transcription factors (1.Marianayagam N.J. Sunde M. Matthews J.M. The power of two: protein dimerization in biology.Trends Biochem. Sci. 2004; 29: 618-625Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Dimerization also has the potential to increase the efficiency and specificity of enzymatic reactions, with more than 70% of enzymes able to self-associate (1.Marianayagam N.J. Sunde M. Matthews J.M. The power of two: protein dimerization in biology.Trends Biochem. Sci. 2004; 29: 618-625Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Dimerization and higher-order oligomerization is also an important and widespread feature among proteins central to membrane trafficking. In this study we focus on a family of proteins, the sorting nexins (SNXs), which regulate vesicular and tubulovesicular transport in the endocytic system (2.Seet L.F. Hong W. The Phox (PX) domain proteins and membrane traffic.Biochim. Biophys. Acta. 2006; 1761: 878-896Crossref PubMed Scopus (163) Google Scholar, 3.Teasdale R.D. Collins B.M. Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions, and roles in disease.Biochem. J. 2012; 441: 39-59Crossref PubMed Scopus (207) Google Scholar). In particular, we examine the propensity for homo- and hetero-association among SNX proteins containing the Bin/Amphiphysin/Rvs (BAR) domain, a family of molecules that utilize dimerization to sense and drive membrane curvature. In vivo studies have shown that the SNX-BAR proteins are localized on tubular and vesicular membrane structures throughout the endocytic network (4.Cullen P.J. Phosphoinositides and the regulation of tubular-based endosomal sorting.Biochem. Soc. Trans. 2011; 39: 839-850Crossref PubMed Scopus (10) Google Scholar, 5.van Weering J.R. Verkade P. Cullen P.J. SNX-BAR proteins in phosphoinositide-mediated, tubular-based endosomal sorting.Semin. Cell Dev. Biol. 2010; 21: 371-380Crossref PubMed Scopus (127) Google Scholar). They have been shown to be involved in a growing array of endosomal sorting events (6.Wang J.T. Kerr M.C. Karunaratne S. Jeanes A. Yap A.S. Teasdale R.D. The SNX-PX-BAR family in macropinocytosis: the regulation of macropinosome formation by SNX-PX-BAR proteins.PLoS One. 2010; 5: e13763Crossref PubMed Scopus (46) Google Scholar, 7.Wassmer T. Attar N. Harterink M. van Weering J.R. Traer C.J. Oakley J. Goud B. Stephens D.J. Verkade P. Korswagen H.C. Cullen P.J. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.Dev. Cell. 2009; 17: 110-122Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 8.van Weering J.R. Verkade P. Cullen P.J. SNX-BAR-mediated endosome tubulation is co-ordinated with endosome maturation.Traffic. 2012; 13: 94-107Crossref PubMed Scopus (113) Google Scholar, 9.Traer C.J. Rutherford A.C. Palmer K.J. Wassmer T. Oakley J. Attar N. Carlton J.G. Kremerskothen J. Stephens D.J. Cullen P.J. SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment.Nat. Cell Biol. 2007; 9: 1370-1380Crossref PubMed Scopus (210) Google Scholar) and both clathrin-dependent and independent endocytosis (10.McGough I.J. Cullen P.J. Clathrin is not required for SNX-BAR-retromer-mediated carrier formation.J. Cell Sci. 2013; 126: 45-52Crossref PubMed Scopus (20) Google Scholar). The 12 members of the SNX-BAR subfamily contain a phox-homology (PX) domain required for membrane association and a C-terminal BAR domain, composed of three α-helices that can dimerize to form a rigid banana-shaped structure (Fig. 1A). The concave surface of the dimeric BAR domains contains basic residues that mediate association with the phospholipid bilayer through electrostatic interactions. These proteins are then able to sense local bending of the membrane, and even drive membrane deformation by forming higher order helical arrays that stabilize high curvature membrane tubules and vesicles (11.Masuda M. Mochizuki N. Structural characteristics of BAR domain superfamily to sculpt the membrane.Semin. Cell Dev. Biol. 2010; 21: 391-398Crossref PubMed Scopus (68) Google Scholar, 12.Qualmann B. Koch D. Kessels M.M. Let's go bananas: revisiting the endocytic BAR code.EMBO J. 2011; 30: 3501-3515Crossref PubMed Scopus (187) Google Scholar, 13.Rao Y. Haucke V. Membrane shaping by the Bin/amphiphysin/Rvs (BAR) domain protein superfamily.Cell. Mol. Life Sci. 2011; 68: 3983-3993Crossref PubMed Scopus (83) Google Scholar). The exact structure of polymerized SNX-BAR proteins has not been elucidated. However, it is known that BAR domain-driven dimerization of the proteins is required. Both homo-dimerization and hetero-dimerization have been observed. For example, and have been shown to form W. Dimerization is required for tyrosine in to signaling and interaction with J. 2006; PubMed Scopus Google Scholar, B. in the BAR domain allow and of sorting nexin J. 2011; PubMed Scopus Google Scholar, J. Y. D. nexin cell and by with Biol. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, and of and sorting nexin Full Text Full Text PDF PubMed Scopus Google Scholar). the other SNX2, and SNX6, which with the retromer trafficking have been to form a of that coat common endosomal membrane tubules M. of a family of sorting nexin molecules and of association with Cell. Biol. PubMed Scopus Google Scholar, Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google Scholar). potential of hetero-dimerization of the SNX-BAR proteins is that formation of allow for of membrane trafficking in a within or of the proteins. Here we a of the in SNX-BAR interaction a combination of cell-free protein expression, proximity and fluorescence of SNX-BAR proteins SNX2, SNX4, SNX6, and the PX SNX protein in a Leishmania cell-free system and for ability to form homo- or in by The homo-dimerization propensity by and by single-molecule used to the of proteins in the SNX the of and and The used are GFP and The SNX proteins into the Gateway Leishmania and the These the and or the D. B.M. P. A.S. B. for of the human One. 2011; PubMed Scopus Google Scholar). the SNX in or with the in the by The Leishmania tarentolae S. W. W. Leishmania cell-free protein 2011; PubMed Scopus Google Scholar). containing of and of for for proteins with by for and on The proteins by the for fluorescence The Assay the and protein in in of of protein to each containing of in and by of in to a of The proteins and for of of by a of in the The the to the for is the of all the for each protein For each the intensity a GFP is in the is The are then to a In this we the for the interaction between and a in all of all the for both of each protein to the GFP and Cherry of SNX proteins in of for The with of the for GFP SNX proteins and of the for a C-terminal SNX GFP used a to the protein a of and the with of coupled with for with by the with of with and The proteins the by for in of and for intensity and a of of the GFP fluorescence into the Cherry for in the of the Y. A. A.C. single-molecule of a protein in Sci. 2009; PubMed Scopus Google Scholar, Y. V. A.C. A. a protein complex by single-molecule 2011; PubMed Scopus Google Scholar). in and in to a of of of each into a that on of a room on a with a For intensity SNX proteins in of of and for in a GFP by a The number of in single-molecule when the intensity a of is the of by a protein complex and to the number of a GFP to the number of GFP in the is by the that proteins through the and that are observed. The the in the and the number of that the proteins can the this is and are more the of an The number of events for each intensity and to the number of events to a by more than is a of intensity per For and Cherry SNX proteins of in of for and in a and a GFP by a fluorescence Cherry by a The fluorescence of the and the number of in single-molecule when the intensity of the a of For each the of the GFP and Cherry for and of the GFP into the Cherry The then the Cherry by the intensity of the In the of Cherry is to and in the of GFP, with are events. The number of events for each and to the number of events to a of single-molecule a of by events per and by for and The the of to events. We to systematically the pairwise interactions between 12 SNX proteins, of all 144 to the number of a cell-free approach used to of to protein while of the We the cell system system of complex human proteins in functional and form W. S. of in and membrane One. 2013; PubMed Scopus Google Scholar). tarentolae is a and the can be generated and S. W. W. Leishmania cell-free protein 2011; PubMed Scopus Google Scholar, S. cell 2009; PubMed Scopus Google Scholar, S. W. the of a Leishmania tarentolae based cell-free One. 2010; 5: PubMed Scopus Google Scholar). The SNX proteins in GFP and proteins. GFP and Cherry fluorescence used to detect proteins and GFP and Collins B.M. Structural and of the Sci. 2010; PubMed Scopus Google Scholar). are and with protein and the ability to in interactions. these we each SNX with an GFP and a C-terminal and each interaction in of the proteins by the of a cell-free with Gateway that to the SNX-BAR a human proteins in and by and can be in the all proteins to proteins be between and and that a with an BAR The human SNX-BAR protein not in these studies is not in is a proximity assay able to detect interactions with a of to T. P. N. M. The of in PubMed Google Scholar, S. S. based on the for cell signaling 2009; PubMed Scopus Google Scholar). a of this assay to the interactions between SNX protein in the cell-free or can even are to and it is a proximity assay to a However, we are an in by proteins the is not and interactions are The on protein (Fig. of the proteins used to the of each The system is for the in protein the protein not have to be feature of the also an interactions not a The in for a of proteins and for a that the can detect interactions between SNX proteins with In the interaction all 144 protein in and for each to the of the of the of the assay we able to it in a We the with the and C-terminal and the a matrix of interactions (Fig. to a which used in We an of of the a for a and then independent of these other these all proteins for SNX1, and SNX5 are able to form homo-dimers, and show for heteromeric In all the with a high of in the we and this interaction is between SNX8 and the domain protein we observe an interaction SNX8 and but association when are We that this is a interaction based on The propensity to form also by a assay (Fig. The proteins with an GFP C-terminal proteins used and by and for GFP and Cherry for in we the of Cherry fluorescence and and the of a of of the and of a of a for interactions and a higher with a interaction by also of the with to the of SNX2, and the of the the of with both interactions of proteins coupled to that to this we to of proteins. is the of proteins and with fluorescent We that GFP and Cherry can be the single-molecule even on proteins Y. N. M. M. A. W. S. Y. N. S. W. Collins B.M. and of on 2013; Google Scholar). The fluorescence intensity of a fluorescent protein can be and used to the number of proteins in protein The SNX-BAR proteins with an GFP in LTE, and the on a for single-molecule For we used a to the GFP of GFP in to to proteins or protein in the proteins through the a fluorescent is The intensity are in the the In the of the intensity be that of a (Fig. In the of higher-order fluorescent proteins through the in a higher However, not all an through the the is not and Y. N. M. M. A. W. S. Y. N. S. W. Collins B.M. and of on 2013; Google Scholar) for In the of the is the of the of that to the form or The which a these used to the and the for the of events which with the propensity of proteins to form In with and SNX1, and SNX5 to that of GFP molecules (Fig. these also a in to the association in but with these show with both and with other members of the family a propensity for or formation that is in all three (Fig. also that is to form or these on a fluorescent and are to the formation of and study we to detect the proteins, with We A. D. to Soc. Trans. 2010; PubMed Scopus Google Scholar, D. S. D. by and of in 2004; PubMed Scopus Google Scholar, D. A. D. S. of human Biol. PubMed Scopus Google Scholar, S. D. studies of Soc. Trans. 2004; PubMed Scopus Google Scholar, N. Y. Y. A. M. W. P. A. W. W. P.J. M. D. of the of and of 2012; Full Text Full Text PDF PubMed Scopus Google Scholar) on of SNX-BAR proteins with GFP and in LTE, the to and to of In single-molecule the are by and for the GFP and Cherry (Fig. is for each in shown in for and for each a of the intensity of Cherry to the intensity of Cherry GFP fluorescence is to that a Cherry protein is in the a to that a GFP protein is and a that both Cherry and GFP are in the a the the proteins with each is to detect between proteins, and the of For each a events are and the are the of events that to each The of a between and an we the homo-dimerization of SNX8 and SNX3 (Fig. we detect a on in the of SNX8 homo-dimers, that with For SNX3 is with The single-molecule also the by for other is for the (Fig. and are for the (Fig. the approach we also the potential SNX8 interaction with the domain protein SNX3 in we find that the proteins in a of molecules SNX8 to of SNX3 The that a of SNX8 can bind a SNX3 but the of this interaction require we the interaction between and Both proteins have high propensity to form on and a interaction between the proteins. is with a that the SNX8 and proteins form heteromeric in a (Fig. of the formation of a than the higher order assembly of and SNX8 not homo- and hetero-oligomerization among of proteins the SNX-BAR family can be and the of in or and are of study is the by S. S. P. that of domains in Biol. 2011; Full Text Full Text PDF PubMed Scopus Google interactions between The study required the and of domains by with fluorescent for on protein cell-free approach the and the study of proteins. it of all protein and we that of the proteins potential The of interactions S. S. P. that of domains in Biol. 2011; Full Text Full Text PDF PubMed Scopus Google a that be by of the that a not be with or with other the domains can and on the In we that of of SNX-BAR proteins not be for all 144 the interaction not be when we and that is required for hetero-dimerization in (Fig. also that between SNX8 and is in a and require assembly for hetero-dimerization to in protein with and proximity assay has been a for M. T. Y. cell-free protein system for 2011; PubMed Scopus Google Scholar). The combination of cell-free protein and has been used to proteins Y. M. T. Y. T. N. S. A. T. Y. Y. T. M. of in of 21: Scopus Google Scholar, M. Y. T. for proteins the protein by cell-free 2010; 9: PubMed Scopus Google Scholar). studies have on a protein and we the of that approach on matrix of interactions. approach fluorescence which information on and protein the Gateway rapid of into that can be in in assay to and a interaction for a human protein and we the specificity of the the of the and the between proteins in a can with the we the interaction matrix in to and In this in other all interactions be both is to the that is to the between to in to or The we used are but this the to for to the of in interactions studies is protein or is important for information the propensity of a protein to is in the and the of is Here we the specificity of interactions in by single-molecule are on proteins, in and The single-molecule and that interactions Here we are able to detect which proteins and the of these interactions. In this study we have applied this approach to the family of human SNX-BAR proteins, which have central roles in endocytic and endosomal membrane trafficking. the SNX-BAR proteins dimerization of the BAR domains is required for both membrane association and the of potential homo- and SNX-BAR is essential for a of we observe the formation of a of both homo- and within the SNX-BAR are in general with studies R.D. Collins B.M. Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions, and roles in disease.Biochem. J. 2012; 441: 39-59Crossref PubMed Scopus (207) Google Scholar, 7.Wassmer T. Attar N. Harterink M. van Weering J.R. Traer C.J. Oakley J. Goud B. Stephens D.J. Verkade P. Korswagen H.C. Cullen P.J. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.Dev. Cell. 2009; 17: 110-122Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google Scholar) for and for but these by examining interactions between all but of the SNX-BAR We that SNX4, SNX6, and form by all three used In van Weering Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google Scholar) the formation of homo- and between of proteins in In with it also that SNX4, and able to form In to and able to in is in to studies of T. Attar N. Harterink M. van Weering J.R. Traer C.J. Oakley J. Goud B. Stephens D.J. Verkade P. Korswagen H.C. Cullen P.J. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.Dev. Cell. 2009; 17: 110-122Abstract Full Text Full Text PDF PubMed Scopus (219) Google and Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google that these proteins not but is by that is able to form in J. J.G. A. T. a novel SNX, with the family of Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). SNX1, and SNX5 not to form is in with by Wassmer and also that both and propensity for T. Attar N. Harterink M. van Weering J.R. Traer C.J. Oakley J. Goud B. Stephens D.J. Verkade P. Korswagen H.C. Cullen P.J. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.Dev. Cell. 2009; 17: 110-122Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). We that in can form high not and that the structure of the BAR domain Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google Scholar). However, it that the BAR domain interaction is to be of the of residues the dimeric The between and the which the proteins are in the tarentolae system the in this of the of the human SNX-BAR proteins. For each the propensity to is for the by by by and by to the of The the in a of the SNX-BAR play an important in the For example, the of SNX1, SNX2, and with each other to regulate retromer endosomal and the is that this formation of SNX-BAR In the SNX1, SNX2, and SNX5 can with other SNX-BAR proteins, T. Attar N. Harterink M. van Weering J.R. Traer C.J. Oakley J. Goud B. Stephens D.J. Verkade P. Korswagen H.C. Cullen P.J. The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network.Dev. Cell. 2009; 17: 110-122Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). interactions that a high in assay have been for example, SNX4 to form with Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google Scholar). that proteins SNX4, SNX6, and can with other in cells also that SNX8 able to all the proteins to a Weering J.R. Traer C.J. D. Cullen P.J. for SNX-BAR-mediated assembly of endosomal sorting J. 2012; PubMed Scopus Google Scholar). this to the of of these proteins by and of the interaction by this is not the that SNX2, SNX4, SNX6, and SNX8 have a propensity to in promiscuous complex of the an interaction of SNX8 with the domain protein show that and bind to each other with a and the in a a and formation of a complex containing molecules of SNX8 and a SNX3 and association is with roles for both SNX8 and SNX3 in endosome to recycling Weering J.R. Verkade P. Cullen P.J. SNX-BAR-mediated endosome tubulation is co-ordinated with endosome maturation.Traffic. 2012; 13: 94-107Crossref PubMed Scopus (113) Google Scholar, J. A. nexin Biophys. 2009; PubMed Scopus Google Scholar, M. I.J. M. M.C. van Weering J.R. van Cullen P.J. Korswagen H.C. retromer transport of the sorting and is required for Cell Biol. 2011; 13: PubMed Scopus Google but the complex require in vivo to functional In we have systematically the of interactions that are between members of the SNX-BAR family in by cell-free for rapid protein and and for of the interactions. of the of a be the of and this be for proteins. The can be in a the protein or that the protein approach not have the of or other protein but it has the potential to a than and We it be a for the of interaction networks N. M. M. Y. cell-free approach to the study of interactions in 2013; PubMed Scopus Google Scholar, M. of protein sorting proteins and assembly into Biol. Cell. PubMed Scopus Google Scholar, P. R.D. and of a sorting nexin to sorting Cell Sci. PubMed Google Scholar, S. but essential of and in the association of retromer with and the trafficking of Cell. Biol. 2007; PubMed Scopus Google Scholar, A. The of sorting nexin J. 2007; PubMed Scopus Google Scholar, N. S. N. S. nexin with and and coordinates Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). with