Research Articles:

Advance online publication

Pavel Simakin1,#, Christian Koch1,# and Johannes M. Herrmann1

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Modular Cloning (MoClo) allows the combinatorial assembly 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 advanced MoClo toolkit that is designed for the baker’s yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting 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 strategy 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 Mo-Clo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.

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AUTHOR CONTRIBUTIONS

C.K. and J.M.H. conceived the project; P.S. and C.K. de-signed, cloned and verified the constructs and strains; P.S. and C.K. carried out the biochemical and microscopic analysis of the expression plasmids and yeast strains and as well as the image analysis; all authors analyzed the data; J.M.H. wrote the manuscript with the help and input of all authors.

ACKNOWLEDGMENTS

We are grateful to John Dueber (UC Berkeley, US) for establishing the MoClo toolkit for yeast (Addgene #1000000061) [14]. This study was supported by the Deutsche Forschungsgemeinschaft (GRK2737 STRESSistance and HE2803/9-2), and the Forschungsiniti-ative Rheinland-Pfalz BioComp.

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© 2023

A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae by Simakin et al. is licensed under a Creative Commons Attribution 4.0 International License.