Split-TurboID enables contact-dependent proximity labeling in cells
Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for curre...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 22; pp. 12143 - 12154 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences
02.06.2020
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Subjects | |
Online Access | Get full text |
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Summary: | Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein–protein interaction or membrane–membrane apposition. At endoplasmic reticulum–mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum–mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by Tony Hunter, The Salk Institute for Biological Studies, La Jolla, CA, and approved April 7, 2020 (received for review November 7, 2019) Author contributions: K.F.C., S.A.C., and A.Y.T. designed research; K.F.C., T.C.B., S.R., T.S., N.D.U., T.T., C.K., H.-W.R., and I.-K.L. performed research; K.F.C. and A.Y.T. analyzed data; and K.F.C. and A.Y.T. wrote the paper. |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.1919528117 |