Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster
Abstract
Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.
Additional Information
© 2021 Stevens et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: October 8, 2020; Accepted: April 12, 2021; Published: May 17, 2021. We thank Winslow Briggs, Arko Dasgupta, Neta Dean, R. Jürgen Dohmen, Jay C. Dunlap, Arlen Johnson, Karim Labib, Jennifer J. Loros, Christof Taxis, Tong-Seung Tseng, Thomas J. Wandless, and Alexander Varshavsky for reagents and/or technical advice. We thank Katie Sieverman, Emily Flynn, and Leslie Dunipace for technical assistance. This work was funded by grants from the NIH Office of the Director (https://www.nih.gov/institutes-nih/nih-office-director) R21OD017964 (awarded to D.S.S., supporting L.M.S., G.K., and D. S.S.), the National Institute of General Medical Sciences (https://www.nigms.nih.gov) R35GM118146 (awarded to A.S., supporting T.K. and A.S.), and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (https://www.nichd.nih.gov) R21HD095639 (awarded to A.S., supporting J.M. and A.S.) of the National Institutes of Health. J.W.S. was funded by the Cell and Molecular Biology Graduate Program and the Institute for Cellular and Molecular Biology at the University of Texas at Austin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Author Contributions: Conceptualization: David S. Stein. Data curation: Leslie M. Stevens, Goheun Kim, Theodora Koromila, David S. Stein. Formal analysis: Goheun Kim, Theodora Koromila. Funding acquisition: Angelike Stathopoulos, David S. Stein. Investigation: Leslie M. Stevens, Goheun Kim, Theodora Koromila, John W. Steele, James McGehee, David S. Stein. Methodology: Leslie M. Stevens, Goheun Kim, Theodora Koromila, John W. Steele, James McGehee, David S. Stein. Project administration: Angelike Stathopoulos, David S. Stein. Resources: Goheun Kim, John W. Steele, James McGehee, Angelike Stathopoulos, David S. Stein. Supervision: Angelike Stathopoulos, David S. Stein. Validation: Leslie M. Stevens, Goheun Kim, Theodora Koromila, James McGehee, Angelike Stathopoulos, David S. Stein. Visualization: Leslie M. Stevens, Goheun Kim, Theodora Koromila, David S. Stein. Writing – original draft: David S. Stein. Writing – review & editing: Leslie M. Stevens, Goheun Kim, Theodora Koromila, James McGehee, Angelike Stathopoulos, David S. Stein. The authors have declared that no competing interests exist.Attached Files
Published - journal.pgen.1009544.pdf
Supplemental Material - journal.pgen.1009544.s001.zip
Supplemental Material - journal.pgen.1009544.s002.zip
Supplemental Material - journal.pgen.1009544.s003.zip
Supplemental Material - journal.pgen.1009544.s004.zip
Supplemental Material - journal.pgen.1009544.s005.zip
Supplemental Material - journal.pgen.1009544.s006.zip
Supplemental Material - journal.pgen.1009544.s007.zip
Supplemental Material - journal.pgen.1009544.s008.zip
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Additional details
- Eprint ID
- 109178
- Resolver ID
- CaltechAUTHORS:20210518-122038067
- NIH
- R21OD017964
- NIH
- R35GM118146
- NIH
- R21HD095639
- University of Texas at Austin
- Created
-
2021-05-19Created from EPrint's datestamp field
- Updated
-
2021-05-27Created from EPrint's last_modified field
- Caltech groups
- Division of Biology and Biological Engineering