Abstract
Molecular switches are molecules that switch between two states to control cellularprocesses. GTPases are a type of molecular switch that induce downstream signaling for many
essential cellular processes through GTP hydrolysis. Rem2 is a GTPase that promotes synapse
formation and decreases dendritic branching through a mechanism that is not well understood.
In order to further elucidate the function of Rem2 we decided to search for Rem2 protein-protein
interactions using the Yeast Two-Hybrid system (Y2H) and a human brain cDNA library. The
Y2H system is a genetic assay that utilizes a modified transcriptional activator from the Gal4
system to screen for protein-protein interactions. In the Y2H system, the Gal4 transcriptional
activator is divided onto two plasmids. Each plasmid contains either the Gal4 activation domain
or binding domain and either a protein of interest (bait) or cDNA from a human library (prey). A
Gal4 promoter is placed in front of reporter genes so that if the protein of interest and a protein
from the library interact, they will induce transcription of the reporter. In our system, Rem2 was
the bait protein and was fused to the DNA binding domain. The prey was a human brain cDNA
library fused to the activation domain. The reporter genes used were HIS3 and ADE2. Samples
containing both the prey and bait plasmids that grew on histidine or adenine dropout plates,
indicating that the human protein they contain interacts with Rem2, were then sequenced and
further processed to confirm their interaction. RhoA, which is a Rho family GTPase, was
determined to interact with Rem2 based on all the tests we performed.