A new method for the selection of protein interactions in mammalian cells

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to ver ify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor kappa B and (2) th e association of Homo sapiens nuclear autoantigen SP100B with human hetero chromatin protein Ice, a protein implicated in chromatin remodelling. We ob serve for the first time that these interactions also occur in vivo. One pr otein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself link ed to guanine phosphoryltransferase 2 (gpt2) modified to begin with an argi nine residue. Upon interaction of both proteins, ubiquitin is reconstituted , and its association with the Rgpt2 reporter is subsequently cleaved off b y ubiquitin-processing enzymes. presence of arginine in the Rgpt2 gene prod uct leads to the degradation of the product by the N-end rule pathway. In t he human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzym e for hypoxanthine-guanine phosphoribosyltransferase) cells in which intera ction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-th ioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.