Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.
We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions.
Fearon
E R
ER
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Finkel
T
T
Gillison
M L
ML
Kennedy
S P
SP
Casella
J F
JF
Tomaselli
G F
GF
Morrow
J S
JS
Van Dang
C
C
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65; D006822:Hybrid Cells; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014764:Viral Proteins