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PUP-IT2 as an alternative strategy for PUP-IT proximity labeling

PUP-IT is a proximity labeling method based on the prokaryotic enzyme PafA. PafA mediates the ligation of Pup, a small peptide, to the proximal proteins. It is different from other proximity labeling methods, such as BioID and APEX, in that both the enzyme and the labeling tag are proteins, which al...

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Detalles Bibliográficos
Autores principales: Yue, Suyu, Xu, Peng, Cao, Zhihe, Zhuang, Min
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9558124/
https://www.ncbi.nlm.nih.gov/pubmed/36250004
http://dx.doi.org/10.3389/fmolb.2022.1007720
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author Yue, Suyu
Xu, Peng
Cao, Zhihe
Zhuang, Min
author_facet Yue, Suyu
Xu, Peng
Cao, Zhihe
Zhuang, Min
author_sort Yue, Suyu
collection PubMed
description PUP-IT is a proximity labeling method based on the prokaryotic enzyme PafA. PafA mediates the ligation of Pup, a small peptide, to the proximal proteins. It is different from other proximity labeling methods, such as BioID and APEX, in that both the enzyme and the labeling tag are proteins, which allows for potential in vivo applications. All proximity labeling involves the genetic fusion of the proximity labeling enzyme with the bait protein. However, PafA is a 55 kDa enzyme which sometimes interferes with the bait function. In this study, we tested an alternative proximity labeling strategy, PUP-IT2, in which only a small 7 kDa protein is fused to the bait protein. We examined the activity of PUP-IT2 in vitro and in cells. We also compared it with the original PUP-IT. Finally, we applied PUP-IT2 coupled mass spectrometry to map protein-protein interactions. Overall, we established a new way to use PUP-IT2 for proximity labeling, and this method may have a broad application.
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spelling pubmed-95581242022-10-14 PUP-IT2 as an alternative strategy for PUP-IT proximity labeling Yue, Suyu Xu, Peng Cao, Zhihe Zhuang, Min Front Mol Biosci Molecular Biosciences PUP-IT is a proximity labeling method based on the prokaryotic enzyme PafA. PafA mediates the ligation of Pup, a small peptide, to the proximal proteins. It is different from other proximity labeling methods, such as BioID and APEX, in that both the enzyme and the labeling tag are proteins, which allows for potential in vivo applications. All proximity labeling involves the genetic fusion of the proximity labeling enzyme with the bait protein. However, PafA is a 55 kDa enzyme which sometimes interferes with the bait function. In this study, we tested an alternative proximity labeling strategy, PUP-IT2, in which only a small 7 kDa protein is fused to the bait protein. We examined the activity of PUP-IT2 in vitro and in cells. We also compared it with the original PUP-IT. Finally, we applied PUP-IT2 coupled mass spectrometry to map protein-protein interactions. Overall, we established a new way to use PUP-IT2 for proximity labeling, and this method may have a broad application. Frontiers Media S.A. 2022-09-29 /pmc/articles/PMC9558124/ /pubmed/36250004 http://dx.doi.org/10.3389/fmolb.2022.1007720 Text en Copyright © 2022 Yue, Xu, Cao and Zhuang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Yue, Suyu
Xu, Peng
Cao, Zhihe
Zhuang, Min
PUP-IT2 as an alternative strategy for PUP-IT proximity labeling
title PUP-IT2 as an alternative strategy for PUP-IT proximity labeling
title_full PUP-IT2 as an alternative strategy for PUP-IT proximity labeling
title_fullStr PUP-IT2 as an alternative strategy for PUP-IT proximity labeling
title_full_unstemmed PUP-IT2 as an alternative strategy for PUP-IT proximity labeling
title_short PUP-IT2 as an alternative strategy for PUP-IT proximity labeling
title_sort pup-it2 as an alternative strategy for pup-it proximity labeling
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9558124/
https://www.ncbi.nlm.nih.gov/pubmed/36250004
http://dx.doi.org/10.3389/fmolb.2022.1007720
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