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 verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein 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 linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.
Rojo-Niersbach
E
E
Max-Delbrück-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linné-Weg 10, 50829 Köln, Germany.
Morley
D
D
Heck
S
S
Lehming
N
N
D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008722:Methods; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D034961:Antigens, Nuclear; D001324:Autoantigens; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D014452:Ubiquitins; C066642:SP100 protein, human