title: Combining Unique Multiplex Gateway Cloning and Bimolecular Fluorescence Complementation (BiFC) for High-Throughput Screening of Protein-Protein Interactions.
abstract: Protein interaction networks are the basis for human metabolic and signaling systems. Interaction studies often use bimolecular fluorescence complementation (BiFC) to reveal the formation and cellular localization of protein complexes. However, large-scale studies were either far from native conditions in human cells or limited by laborious restriction/ligation cloning techniques. Here, we describe a new tool for protein interaction screening based on Gateway-compatible BiFC vectors. We made a set of four new vectors that permit fusion of candidate proteins to the N or C fragment of Venus in all fusion positions. We have validated the vectors and confirmed self-association of AHCY, AHCYL1, and galectin-3. In a high-throughput BiFC screen, we identified new AHCY interaction partners: galectin-3 and PUS7L. We also describe additional steps in protein interaction analysis, applied for AHCY-galectin-3 interaction. First, we classified the interaction in intracellular vesicles using CellCognition, machine learning free software. Then we identified the vesicles as endosomal pathway compartments, in line with known galectin-3 trafficking route. This offers a platform to rapidly identify and localize new protein interactions inside living cells, a prerequisite to validate in silico interactome data, and ultimately decode complex protein networks.
authors.affiliation: 1 Department for Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Adriana
authors.initials: A
authors.lastname: Lepur
authors.affiliation: 1 Department for Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Lucija
authors.initials: L
authors.lastname: Kovačević
authors.affiliation: 1 Department for Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Robert
authors.initials: R
authors.lastname: Belužić
authors.affiliation: 1 Department for Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.
authors.forename: Oliver
authors.initials: O
authors.lastname: Vugrek
mesh_terms: D043383:Adenosylhomocysteinase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D019295:Computational Biology / Q000379:methods; D037502:Galectin 3 / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism; D037181:Lectins, C-Type / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps / Q000235:genetics*
chemical_list: C470184:CLECL1 protein, human; D037502:Galectin 3; D037181:Lectins, C-Type; D008565:Membrane Proteins; D043383:Adenosylhomocysteinase; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase
keywords: AHCY; AHCYL1; BiFC; Gateway; galectin-3; high throughput