Tool on Protein Translocation
2023/10/12
M2 Hiroki Umeda

1Outline

2

1. Introduction

2. Latest Finding
1. Tools to analyze protein translocation
2. Tools to control protein translocation

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Introduction: Protein localization
Localization:

・Many proteins are transported to specific organelles where they work.
・This localization is achieved mainly by 2 trafficking systems.
① Signal Sequence (gray arrow)・nuclear localization signal (NLS)・mitochondrial matrix targeting signal (MTS)・ER signal sequencesetc.

② Vesicular Transport (orange arrow)・Rab / tethering protein+ v-SNARE / t-SNARE interaction

Genereux, J. C. et al. ChemPlusChem 2021, 86, 1397.

These trafficking systems sequester proteins into compartments,
thereby allowing parallel processing of various reactions.

4Introduction: Protein Translocation
Protein localization is NOT static event→ Protein Translocation :proteins localization is dynamically regulated in response to external stimuli or cellular state
Case 1)Case 2)

Ro, S. W. et al. Cancers 2021,13, 3026.

Hirata,T. et al. 生化学 2018, 90, 125.
Waldmann, H. et al. Nat. Chem. Biol. 2006, 2, 518.

Protein translocation are closely related to cellular function→ Chemical tools on analyze or control protein translocation is important.

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Today’s Contents① TransitID② SLIPT

As Tools to analyze protein translocation

As Tools to control protein translocation

Ting, A. Y. et al. Cell 2023, 186, 3307.

Tsukiji, S. et al. Cell Chem. Biol. 2022, 29, 1446.

Outline

6

1. Introduction

2. Latest Finding
1. Tools to analyze protein translocation
2. Tools to control protein translocation

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Conventional Method① Immunofluorescence (IF)✔ No genetic manipulation✘ Fixation of tissue✘ Non-specificity and availability of some antibodies✘ Small number of proteins observed at once

Taken from “ibidi” website(https://ibidi.com/content/364-the-principle-of-immunofluorescence-assays)② Genetically engineered fusions with fluorescent proteins✔ Direct observation over time
✘ Genetic manipulation✘ Possible effects of fused protein on target protein✘ Small number of proteins observed at once
Garcia-Manyes, S. et al. Nat. Phys. 2019, 15, 973

Challenge: No method for analyzing protein tranlsocation without bias.

This Time’s Method: “TransitID”
TransitID (Trafficking Analysis by Sequential Incorporation of Tags for Identification)
・Tool for unbiased analysis of protein translocation.
・Proximity Labeling (PL) with different methods (TurboID, APEX)
at each of two points where translocation is to be observed.

Ting, A. Y. et al. Cell 2023, 186, 3307.

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9Proximity Labeling (PL)Proximity Labeling (PL)Random labeling of proteins in proximity to the protein of interest (POI)
for the purpose of protein-protein interaction analysis, etc.
APEX2TurboID

Ting, A. Y. et al. Nat. Biotechnol. 2018, 36, 880.

Zou, P. et al. Curr. Opin. Chem. Biol. 2021, 60, 30.
Taken from “wikipwdia” website
Kehlenbach, R. H. et al. J. Biol. Chem. 2019, 294, 16241

10Procedure of TransitID
Basic Procedure① Designate two points to be analyzed fortranslocation② Express TurboID at the start point
and APEX2 at the end point③ Add biotin before translocation
for biotin labeling at the start point

Orthogonal④ Wait for translocation⑤ Add H2O2 and alkyne-conjugated phenol
for alkyne labeling at end point⑥ Perform click reaction to convertalkyne to fluorescein in the lysate⑦ Proteins dual-labeled withbiotin and fluorescein are enrichedby sequential purification⑧ Analyze with mass spectrometry

Dual Labelled Proteins||Translocated Protein !
Ting, A. Y. et al. Cell 2023, 186, 3307.

11Validation of TransitID
Detection of protein translocationfrom cytoplasm to mitochondria
Start point: Turbo-ID NES @ cytoplasm
End point: APEX2-mito @ mitochondrial matrix
・Compared to proteins labeled by APEX2,proteins dual labeled by APEX2 and turboID
were less abundant in SDS-PAGE (fig. C).
・Proteins that were NOT visible
after the second purification were proteinsderived from mitochondrial DNA (fig. D)
・Proteins that were visible
after the second purification were proteinsderived from nuclear DNA (fig. D)labeled by
APEX2

Also labeled by
TurboID

Ting, A. Y. et al. Cell 2023, 186, 3307.

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Application: Translocation under Stress
Translocation under Stress
Case1: Hikeshi-Hsp70 translocation

Case2: Stress Granule (SG) formation

Analyze

Ivanov, P. et al. Biochim. Biophys. Acta
Mol. Cell. Res. 2011, 1868, 118876.

Imamoto, N. et al. Cell 2012, 149, 578.
Ting, A. Y. et al. Cell 2023, 186, 3307.

Application: Translocation under Stress

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Detection of protein translocationfrom Nucleolus/Nucleus to SG
Start point: LOV-Turbo-G3BP1 @SG
End point: APEX2-NIK3x @Nucleolusor APEX2-NLS @Nucleus

Ting, A. Y. et al. Nat. Methods
2023, 20, 908.

(Comparison of blue and red samples)・20 proteins were identified that translocatefrom SG to nucleolus
・1 protein (JUN) was identified that translocatefrom SG to nuclear (other than the nucleolus).

→ Many of known SG proteins were identified,
supporting the reliability of the results.
→ Unreported SG proteins were also identified.
(Comparison of blue and light-blue samples)

・Time scale of translocation differs among proteins.
Ting, A. Y. et al. Cell 2023, 186, 3307.

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Validation: Is JUN truly unreported SG proteins ?
Validation of whether JUN is SP protein

The role of JUN under stress・The observation of fusions with photoactivatable GFP (paGFP)
indicates that JUN is an SG protein・JUN translocation to SG during stress suppress its aggregation.
・It enables JUN to quickly re-localizes to the nucleus
and get active after stress.
Ting, A. Y. et al. Cell 2023, 186, 3307.

Summary and Outlook

15・Ting et al. have developed a general method
for unbiased analysis of proteins translocationin an arbitrary spatio-temporal setting: “TransitID.”
・This was accomplished using an orthogonal proximity labelingwith modified APEX2 and TurboID.

・This method was successful in identifying a variety of proteins,
including JUN, whose localization has not been known before.
・This method can also be used to analyze protein translocation
between cells (omitted this time).

・This method inevitably inherits the issues of parental methods.
Turbo ID : low resolution due to long labeling time (10-60 min.)
APEX2 : toxicity of H2O2・Development of improved proximity-labeling enzymes
also improve this method.

Ting, A. Y. et al. Cell 2023, 186, 3307.

Outline

16

1. Introduction

2. Latest Finding
1. Tools to analyze protein translocation
2. Tools to control protein translocation

1

Conventional Method: CID

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Artificial control of the protein translocation

Useful as a means of elucidating the function of the protein

Chemically-Inducible Dimerization (CID)
A method of dimerizing two proteins,with an organic molecule (dimerizer)
1st generation;
FKBP12/FRB dimerization with Rapamycin✔ Versatility✘ Binding to endogenous FKBP12 and mTOR
✘ Almost irreversible binding (Kd=200 pM)
Inoue, T. et al. Pflugers. Arch. 2013, 465, 409.

Conventional Method: Improved CID

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Challenge 1: Bio-orthogonality
・F36V mutant of FKBP12 and SLF' ligands for it

・Use of different types of tags such as
Halo, SNAP, BL tags, etc.

taken form "Bloomington Drosophila Stock Center" website
Mizukami, S. et al. Angew. Chem. Int. Ed. 2021, 60, 11378.

Challenge 2: Reversibility
Combination of TMP and eDHFR・Competitively dissociates dimerizers
by adding an excess of free TMP・Small concern for bio-orthogonality(for eDHFR; KI=1nM vs. for mDHFR; KI=4~8µM)

Wu, Y. et al. Angew. Chem. Int. Ed.
2014, 53, 10049.

19This Time’s Method: ”SLIPT”
Intrinsic problems with CID✘ Strict concentration control of dimerizers is required.
✘ Genetic manipulation is performed on two or more proteins.

SLIPT (Self-localizing Ligand-Induced Protein Translocation)

CID :Localized
protein

POITagSLIPT :

dimerizer

Self-localizing ligand

・Localization is controlled by 1-to-1 bindingof the protein of interest (POI)
to the self-localizing ligand

・Self-localizing ligands (SL)= tag protein ligand + localization motife.g.) TMP for eDHFR

tag protein ligande.g.) Hoechst for nucleus
Taxol for microtubule

localization motif
Tsukiji, S. et al. J. Am. Chem. Soc.
2013, 135, 12684.

Tag

20SLIPT-PM
SLIPT-PM・SLIPT system to translocate POI to the plasma membrane (PM)・translocation to the PM is achieved palmitoylated mgc motif

ligandmgc motif(localization motif)

Problem 1) Transient localization
Problem 2) Low selectivity of localization
Tsukiji, S. et al. J. Am. Chem. Soc. 2013, 135, 12684.
Tsukiji, S. et al. ACS Chem. Biol. 2020, 15, 837.

Peterson, B. R. et al. J. Am. Chem. Soc. 2002, 124, 2444.

Improved Selectivity in SLIPT-PM (Solution to Problem 1)
Problem:Transient localization to the PM (plasma membrane)
Cause:
Cleavage of amide bond between Cys in mgc motif and linker in cell
Solution:
Modification of the motif to a more degradation-resistant motif:
・Conversion of L-Cys to D-CysmDc motif is the best
・Removal of Gly

Tsukiji, S. et al. ACS Chem. Biol. 2020, 15, 837.

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Improved Persistence in SLIPT-PM (Solution to Problem 2)
Problem:Localized to the Golgi surface as well as the PM (plasma membrane)
Cause:
Rapid depalmitoylation reaction
Solution:
Reduces the rate of dissociation from the PM→ Add a domain to the tag proteinthat enhances its interaction with the PM= Cationic Lys residues; K6 is the best

Tsukiji, S. et al. ACS Chem. Biol. 2020, 15, 1004.

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Latest Version of SLIPT-PM
Latest Version of SLIPT-PM
Self-localizing ligands: TMP with mDc motif
Tag fused to POI: eDHFR with K6 grafted between D69 and D70; iK6DHFR
more versatile tag that can fuse proteins to the N- as well as the C-terminus of eDHFR(Previous version cannot fuse protein at the C-terminus.)

Optimized SLIPT-PM

Application

Tsukiji, S. et al. Cell Chem. Biol. 2022, 29, 1446.

Application: Multiplex Control of Signal Transduction

24Observation of the interaction of two signaling pathways① Add mgcBCP → Activation of Ras/Erk pathway(No activation of PI3K/Akt pathway)② Add mgcTMP → Activation of PI3K/Akt pathway→ Activation of Ras/Erk pathway ! (positive feedback)PI3K subunits

Tsukiji, S. et al. ACS Chem. Biol. 2020, 15, 1004.

Application: Induction of Signal Oscillation
Procedure for Signal Oscillation

・ Reversible binding of TMP-eDHFR・ Strong binding of mgc motif - PM

① Add mDcTMP→ Raf translocation to PM leading to ERK activation (Nucleus translocation)② Add free TMP→ Raf dissociation from PM
leading to ERK deactivation(Cytoplasm translocation)③ Wash (Remove free TMP)
→ Raf re-translocation to PM
leading to ERK activation(Nucleus translocation)
Repetition of ② and ③
induce signal oscillation(≈ repetitive activation)
Tsukiji, S. et al. Cell Chem. Biol. 2022, 29, 1446.

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Summary and Outlook
・Tsukiji et al. have developed a useful tool
to control proteins translocation (especially to PM): “SLIPT(-PM).”・Latest tags; iK6DHFR are universal and can be inserted
in a variety of positions, including inside proteins
・SLIPT was successful in multiplex or repetitive activationof signaling pathways.

・SLIPT systems are slower than CID systems(t1/2 in SLIPT; 2.6~6.1min. vs. t1/2 in CID; 20~50 sec.)
・localization motif is limited to a few types.
→ Vigorous search for localization motifs will expand the versatility.
cf.) Other methods of protein localization to the PM; GCEPeng, T. et al. Chem. Commun. 2020, 56, 13880.

Tsukiji, S. et al. Cell Chem. Biol. 2022, 29, 1446.

Lin, S. et al. Nat. Chem. Biol. 2023,
https://doi.org/10.1038/s41589-023-01400-8.

27Translocation in Disease
Disruption of protein translocation leads to several disease.
Case 1 ) Cancer

Li, S. et al. Biochim. Biophys. Acta Rev. Cancer 2014, 1846, 13.

Case 2 ) ALS

Onodera, O. et al. 臨床神経, 2020, 60, 109.

Tools to normalize protein mislocalization can be therapeutic.

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Application to Therapy
Related Works
Translocation of endogenous proteins→ However, regulated protein is specific ones used as tags.
Case 1) SLIPT

Case 2) CID

Tsukiji, S. et al. J. Am. Chem. Soc. 2013, 135, 12684.

Challenge

Need small Kd ↓Schreiber, S. L. et al. bioRxiv, 2023.
https://doi.org/10.1101/2023.07.07.548101.

↓ inhibitor cannot be used

Difficulty in developing ligands that can translocate POI without impairing its function→ Covalently introduce localization motifs at sites with minimal effect on POI (with LDC) ?

Thank you for your kind attention.

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