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

Authors:

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

doi: 10.15698/mic2023.04.794
Volume 10, pp. 78 to 87, published 28/02/2023.

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Affiliations:

1 Cell Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany.
# Both authors contributed equally.

Keywords: 

expression plasmids, mitochondrial import, modular cloning, promoter strength, protein targeting, split GFP.

Corresponding Author(s):

Johannes M. Herrmann, Cell Biology, University of Kaiserslautern, Erwin-Schrödinger-Strasse 13, 67663 Kaiserslautern, Germany; Phone: +49 6312052406; hannes.herrmann@biologie.uni-kl.de

Conflict of interest statement:

The authors declare no competing financial interests.

Please cite this article as:

Pavel Simakin, Christian Koch and Johannes M. Herrmann (2023). A modular cloning (MoClo) toolkit for reliable intra-cellular protein targeting in the yeast Saccharomyces cerevisiae. Microbial Cell 10(4): 78-87. doi: 10.15698/mic2023.04.794

© 2023 Simakin et al. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Abstract:

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.