Array MAPPIT: high-throughput interactome analysis in mammalian cells.
Physical interactions between proteins play a key role in probably every cellular process. Efforts to chart the protein interaction networks are ongoing in a number of model organisms using a diversity of approaches. The resulting genome-wide interaction maps will provide a scaffold for further detailed functional analysis. We developed MAPPIT, a mammalian two-hybrid approach that allows identification and analysis of mammalian protein-protein interactions in their native environment. Here, we introduce an efficient MAPPIT assay that permits high-throughput screening of arrayed collections of proteins and complements a previously published cDNA library screening approach. We validated both methods in screens for interaction partners of the Cullin-based E3 ubiquitin ligase subunits SKP1 and Elongin C. In addition to a number of known interactors, novel SKP1 and Elongin C binding proteins were identified. The array assay is an important addition to the MAPPIT suite of technologies that is expected to significantly increase its utility as a toolbox to screen for novel interactors of proteins or small molecules.
Lievens
Sam
S
Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium.
Vanderroost
Nele
N
Van der Heyden
José
J
Gesellchen
Viola
V
Vidal
Marc
M
Tavernier
Jan
J
D000818:Animals; D002460:Cell Line; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D020798:Two-Hybrid System Techniques
D011506:Proteins