New genetic system for translocating proteins through the mitochondrial membrane of the yeast Yarrowia lipolytica

Abstract

Currently, the extremophilic yeast Yarrowia lipolytica is used as a powerful system for expression of recombinant proteins. However, attempts to obtain recombinant producers based on the Y. lipolytica yeast with cytosolic location of the product almost always fail. It concerns proteins with N-terminal sequences, which are detected by proteasomes and induce the product proteolysis. For stabilization of recombinant products in the Y. lipolytica cells, they should be transported from cytoplasm into mitochondria, where they will be shielded from ubiquitination and from contact with proteosomes. In this work, a method is designed that enhances the proteolytic stability of recombinant proteins in Y. lipolytica without N-terminal modification. For the protein transport through the mitochondrial membrane of Y. lipolytica, a genetic system is proposed that employs specific non-translated sequences of mRNA and performs cotranslational targeting of proteins into mitochondria. Using a fluorescent protein 6His-EYFP that has a low stability in Y. lipolytica cytoplasm as a model, it has been shown that gene expression of this protein under the control of the promoter and transcriptional terminator of mitochondrial superoxide dismutase SOD2 leads to the accumulation in the yeast mitochondria of fluorescent product displaying a high stability in vivo. A new system of targeting recombinant proteins into mitochondria can be of great importance for pharmaceutical purposes, as it enables the creation of proteins with a native primary structure without leader sequence. This system can be used to create a genetic system for maintenance and modification of human native mitochondrial genome in vivo and for the treatment of genetic mitochondrial illnesses.