Description:
<jats:p>Analysis of membrane protein interactions is difficult because of the
hydrophobic nature of these proteins, which often renders conventional
biochemical and genetic assays fruitless. This is a substantial problem
because proteins that are integral or associated with membranes represent
approximately one-third of all proteins in a typical eukaryotic cell. We have
shown previously that the modified split-ubiquitin system can be used as a
genetic assay for the in vivo detection of interactions between the two
characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called
split-ubiquitin membrane yeast two-hybrid (YTH) system uses the
split-ubiquitin approach in which reconstitution of two ubiquitin halves is
mediated by a protein–protein interaction. Here we converted the
split-ubiquitin membrane YTH system into a generally applicable in vivo
screening approach to identify interacting partners of a particular mammalian
transmembrane protein. We have demonstrated the effectiveness of this approach
by using the mammalian ErbB3 receptor as bait and have identified three
previously unknown ErbB3-interacting proteins. In addition, we have confirmed
one of the newly found interactions between ErbB3 and the membrane-associated
RGS4 protein by coimmunoprecipitating the two proteins from human cells. We
expect the split-ubiquitin membrane YTH technology to be valuable for the
identification of potential interacting partners of integral membrane proteins
from many model organisms.</jats:p>