A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae

Modular Cloning (MoClo) allows the combinatorial as-sembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an ad-vanced MoClo toolkit that is designed for the baker's yeast Saccha-romyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different tar-geting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strate-gy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.