Using GBP Nanotrap to Restore Autophagy in the Rab5/Vps21 Mutant by Forcing Snf7 and Atg17 Interaction

Abstract

Protein–protein interactions are important for physiology performance. Green fluorescent protein (GFP) is a widely used protein tag to show protein localization in vivo. GFP binding protein (GBP) is a specific domain with high affinity to GFP. A novel technique with GBP fused protein X tagged with red fluorescence protein binding to GFP of GFP fused protein Y to establish a close association for proteins X and Y independently from other proteins has recently been developed. It is found that the interaction and colocalization between Snf7 and Atg17 is impaired in Saccharomyces cerevisiae vps21Δ cells, which are defective in autophagy. In order to determine whether the interaction between Snf7 and Atg17 is important for autophagy, we forced the interaction between Snf7 and Atg17 through GBP–GFP binding. Snf7-GBP-mCherry and/or GFP-Atg17 tagged wild-type and vps21Δ cells were compared for autophagy process under starvation by determining the maturation of proprotein of Ape1 (prApe1). Our results showed that the defective autophagy in vps21Δ cells was significantly suppressed when both Snf7-GBP-mCherry and GFP-Atg17 were installed. Our results indicate that the GBP–GFP nanotrap technique is a powerful tool to restore colocalization/interaction in vivo and the Snf7–Atg17 interaction is important for yeast autophagy.