Journal of the American Chemical Society
Communication
(Figure 3a). Immunostaining and co-IP experiments (Figure S24
and S25) similarly indicated that the reporter protein moved to
Golgi and then reverted to ER when ALiS was washed out,
whereas no reversibility was observed with biotin. In vitro SPR
experiments (Figure S26) also supported the occurrence of these
processes. Finally, although we cannot rule out the presence of
off-target proteins in cells, ALiS showed no cytotoxicity in the
concentration range used for imaging (Figure S27) and induced
no morphological change or other distinctive behavior of HeLa
cells, which implies that these compounds do not interfere with
critical cell signaling.
22000006 to T.N., 25286051 and 25650051 to S.S., and Platform
for Drug Discovery, Informatics, and Structural Life Science), as
well as The Mochida Memorial Foundation for Medical and
Pharmaceutical Research (to T.T.). Work at the F.P. lab has been
supported by Institut Curie, CNRS, and by grants from the
́
Fondation pour la Recherche Medicale (DEQ20120323723),
and from the Agence Nationale pour la Recherche (ANR-12-
BSV2-0003-01). A part of this work was performed at the SPring-
8 BL38B1 and BL44XU beamlines with the approval of JASRI
(Proposals 2012A1370, 2012B6724, 2012B1295, and
2013A6827).
In conclusion, our large-scale screening system identified
novel streptavidin ligands (ALiS) with Kd values of approx-
imately 1 μM. In conjunction with the SBP tag, their fast koff
(>0.1 s−1) makes them unique tools as competitive ligands, as
illustrated herein by their application to reversibly control
protein transport between ER and Golgi. This approach, for
example, would be applicable to transiently activate post-
translational protein modification, induce reversible signaling,
or reversibly alter protein localization in cells. It will also enable
kinetic studies to identify compounds/genes that specifically
control forward/backward transport of proteins of interest. A
straightforward extension of this work would be vesicle-
independent reversible control of protein translocation from
cytosol to a particular organelle. A few such systems using
FKBP17 and tobacco 14-3-318 have been reported recently, but
we use a simpler ligand (i.e., a readily derivatizable synthetic
compound) and a shorter peptide (SBP) than previous systems.
Importantly, our system is orthogonal to other methods. We are
currently optimizing ALiS in terms of solubility, affinity, and
binding to streptavidin mutants7 to pave the way for further
applications.
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Corresponding Authors
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Yukio Tada and Hirofumi Nakano for helpful advice
on derivative synthesis, Hiroki Sakamoto for help with DNA
work, Hirotatsu Kojima and other staff of DDI, The University of
Tokyo, for advice on screening, Kazuko Oka and Jun Tamura for
protein ESI-MS analysis, and Hanako Ishida for X-ray
crystallography. We also thank Vincent Fraisier from the
PICT-IBiSA Lhomond imaging facility at Institut Curie. This
research was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology of Japan (Grant-in-Aid
for Scientific Research, Grants 23651231 and 25104506 to T.T,
V.; Stein, F.; Rutkowska, A.; MacNamara, A.; Depner, S.; Klingmuller,
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U.; Saez-Rodriguez, J.; Schultz, C. Angew. Chem., Int. Ed. 2014, 53, 6720.
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U. S. A. 2013, 110, E377.
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX