Development of a SNARE-based cell fusion system in Pichia pastoris

Abstract

SNAREs are the main parts of the fusion machinery in synaptic vesicles in mammalian cells. The main fusion proteins involved are VAMP (or synaptobrevin), also known as the v-SNARE, and the complex made up of SNAP-25 and syntaxin as the t-SNARE. It was previously shown that flipped SNAREs can also be used to fuse two mammalian cells. In this study, we modified this system with the aim of generating cell fusion between two Pichia pastoris yeast cells. The mutated SNAREs proteins, as reported by (Hu et al. 2003), were used and were codon-optimized for expression in P. pastoris. The outward orientation was achieved using the alpha-factor, which was fused to the N-terminus of each SNARE mediating protein. A strain expressing flipped VAMP2 carries a cytosolic DsRed and the second strain expressing flipped syntaxin1, flipped SNAP-25, and a secreted complex of VAMP2 was created, expressing a nuclear GFP. Yeast spheroplasts were generated after harvesting in the exponential growth phase and a Zymolase treatment. Spheroplasts of strains with compatible SNARE systems were incubated together, and the fusion events were visualized by fluorescence microscopy. After 3 hours, a fusion efficiency of about 16% was obtained. The fused cells were recovered on plates containing both dominant selection markers. In conclusion, we showed that P. pastoris spheroplasts can be fused using a SNARE mediating procedure. This system can be further developed as a tool for cross-species hybridization (e.g., Pichia spp. & Saccharomyces spp.) which enables the implementation of different pathways in a yeast host cell.