Detection of protein-protein interactions through vesicle targeting

Abstract

The detection of protein-protein interactions through two-hybrid assays has revolutionized our understanding of biology. The remarkable impact of two-hybrid assay platforms derives from their speed, simplicity, and broad applicability. Yet for many organisms, the need to express test proteins in Saccharomyces cerevisiae or Escherichia coli presents a substantial barrier because variations in codon specificity or bias may result in aberrant protein expression. In particular, nonstandard genetic codes are characteristic of several eukaryotic pathogens, for which there are currently no genetically based systems for detection of protein-protein interactions. We have developed a protein-protein interaction assay that is carried out in native host cells by using GFP as the only foreign protein moiety, thus circumventing these problems. We show that interaction can be detected between two protein pairs in both the model yeast S. cerevisiae and the fungal pathogen Candida albicans. We use computational analysis of microscopic images to provide a quantitative and automated assessment of confidence.

Figure 1.—

VCI assay for S. cerevisiae Snf1:Snf4 and Pbs2:Hog1. An S. cerevisiae vps4Δ strain was transformed with a Snf1-GFP fusion plasmid and either a Vps32 vector (A) or a Snf4-Vps32 fusion plasmid (B). The vps4Δ strain was independently transformed with a Pbs2-GFP fusion plasmid and either a Vps32 vector (C) or a Hog1-Vps32 fusion plasmid (D). GFP fluorescence images are shown.

Figure 2.—

Comparison of GFP localization and FM4-64 staining. An S. cerevisiae vps4Δ mutant host expressing both Snf1-GFP and Snf4-Vps32 was stained with membrane dye FM4-64. The dye accumulates in endosome-derived class E compartments in vps4 mutants. DIC, GFP, and FM4-64 channels are shown. White arrows indicate regions of GFP and FM4-64 colocalization.

Figure 3.—

VCI assay for C. albicans Snf1:Snf4 and Pbs2:Hog1. A C. albicans vps4Δ/vps4Δ strain was transformed to introduce a Snf1-GFP fusion gene and either the Vps32 vector (A) or a Snf4-Vps32 fusion gene (B). The vps4Δ/vps4Δ strain was independently transformed to introduce a Pbs2-GFP fusion gene and either the Vps32 vector (C) or a Hog1-Vps32 fusion gene (D). GFP fluorescence images are shown.