A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast

Authors:

Rossella Avagliano Trezza1,#, Janny van den Burg1, Nico van den Oever1 and Ben Distel1,2

doi: 10.15698/mic2018.03.620
Volume 5, pp. 150 to 157, published 05/12/2017.

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Affiliations:

1 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

2 Department of Neuroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

# Current Address: Department of Neuroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

Keywords: 

ubiquitination, sumoylation, modular vectors, inducible expression, yeast, Saccharomyces cerevisiae

Corresponding Author(s):

Ben Distel, PhD, Academic Medical Center, University of Amsterdam, Department of Medical Biochemistry; ErasmusMC, Department of Neuroscience, Wijtemaweg 80, 3015 CN Rotterdam; Phone: (+31) 010 7043571; b.distel@amc.uva.nl

Conflict of interest statement:

The authors declare no conflict of interest.

Please cite this article as:

Rossella Avagliano Trezza, Janny van den Burg, Nico van den Oever and Ben Distel (2017). A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast. Microbial Cell 5(3): 150-157. doi: 10.15698/mic2018.03.620

© 2017 Avagliano Trezza 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:

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex post-translational modifications in a cellular setting.