Quenching Accumulation of Toxic Galactose-1-Phosphate as a System to Select Disruption of Protein-Protein Interactions in Vivo

Abstract

The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.

Current Experimental Methods for Characterizing Protein–Protein Interactions

“…(7) Reverse two‐hybrid system is an upside‐down version of Y2H, in which disruption of the bait–prey interaction decreases the expression of a counter‐selectable reporter gene, such as URA3, CYH2 and GAL1, and allows cell growth under selective conditions. This method facilitates the detection of dissociation of PPIs, and the selection of small‐molecule inhibitors against PPIs that could be developed as therapeutic agents …”

A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype

“…The actions of GALK1 and GALT maintain the level of galactose-1-phosphate. The increase of galactose-1-phosphate, by an abnormal metabolic pathway, may cause various problems, as galactose-1-phosphate is thought to be a toxic metabolite [ 18 ]. Classic galactosemia is a representative disease of an accumulation of galactose-1-phosphate, by GALT deficiency, which is known to be the most common cause of galactosemia [ 19 ].…”

High-Throughput Screening Assays to Discover Small-Molecule Inhibitors of Protein Interactions

“…In a GAL7 null background, GAL1 expression causes accumulation of galactose-1-phosphate, which eventually kills yeast. Pilot assays (including a pilot screening) have shown that this system could be used to detect small-molecule inhibitors of protein interactions, with a good sensitivity [36]. In contrast with URA3 or CYH2 reporters, this GAL1 system does not necessitate the use of a small molecule to achieve toxicity.…”