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A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

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Date
12/2017
Author
Hinrichsen, M.
Lenz, M.
Edwards, J. M.
Miller, O. K.
Mochrie, S. G. J.
Swain, P. S.
Schwarz-Linek, U.
Regan, L.
Keywords
Protein engineering
Fluorescence imaging
Post-translational labeling
QD Chemistry
QH301 Biology
Biochemistry
NDAS
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Abstract
We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein’s turnover time from such data.
Citation
Hinrichsen , M , Lenz , M , Edwards , J M , Miller , O K , Mochrie , S G J , Swain , P S , Schwarz-Linek , U & Regan , L 2017 , ' A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking ' , Protein Engineering, Design & Selection , vol. 30 , no. 12 , pp. 771-780 . https://doi.org/10.1093/protein/gzx059
Publication
Protein Engineering, Design & Selection
Status
Peer reviewed
DOI
https://doi.org/10.1093/protein/gzx059
ISSN
1741-0126
Type
Journal article
Rights
© The Author 2017. Published by Oxford University Press. This work has been made available online in accordance with the publisher’s policies. This is the author created, accepted version manuscript following peer review and may differ slightly from the final published version. The final published version of this work is available at https://doi.org/10.1093/protein/gzx059
Description
This work was funded by the Raymond and Beverley Sackler Institute for Biological, Physical, and Engineering sciences [to L.R.]; the National Institute of Health [Grant nos. GM118528 and CA209992 to M. H. and L. R.]; the Medical Research Council [Grant no. MR/K001485 to U.S.L. and J. M. E.]; a Leverhulme Trust Visiting Professorship [to L. R.]; and Royal Society of Edinburgh [Caledonian Scholarship to O.K.M.].
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  • University of St Andrews Research
URL
https://academic.oup.com/peds/article/30/12/771/4710354#supplementary-data
URI
http://hdl.handle.net/10023/16634

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