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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Abstract

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Introduction

A vast amount of genomic information remains uninterpretable due to our incomplete understanding of cellular interactomes. Conventional protein-protein interaction detection methodologies such as protein co-immunoprecipitation with/without chemical cross-linking and protein co-localization, though widely used, pose a range of disadvantages. Some  of the main disadvantages include poor quantification of the interactions and the potential introduction of non-native binding events. In comparison, emerging proximity-based techniques provide an alternative and a powerful approach for capturing protein interactions in cells. The proximity ligation assay (PLA)

Protocol

1. HeLa Cell Preparation

  1. Prepare the following materials: PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.4; DMEM, supplemented with 10% FCS and 1% Pen-Strep; 4% paraformaldehyde in PBS; 0.5% Triton-X100 in PBS; TBS-T (150 mM NaCl, 20 mM Tris, 0.05% Tween), pH 7.4; 0.0001% sterile poly-L-Lysine solution; 10-well diagnostic slides; a humid chamber; tissue paper; and Coplin slide-staining jars.
    NOTE: To ensure optimal fixation.......

Representative Results

Our previous in vitro studies using purified proteins revealed that a subset of human class A and class B JDPs form transient mixed class JDP complexes to efficiently target a broad range of aggregated proteins and possibly facilitate the assembly of Hsp70-based protein disaggregases7. We employed PLA to determine whether mixed class (A+B) JDP complexes occur in human cervical cancer cells (HeLa). Human JDPs DNAJA2 (class A) and DNAJB1 (class B) were targeted with highly specific primary anti.......

Discussion

Co-immunoprecipitation and co-localization based approaches have been used as long-standing methods to characterize protein assembles. The detection of transiently formed specific chaperone complexes is a major challenge with such conventional methods, and as a result, previous findings are largely restricted to qualitative  interpretations. The cell lysis-based co-immunoprecipitation techniques often require cross-linking to stabilize protein-protein interactions. Cell lysis increases the risk of disrupting tr.......

Acknowledgements

NBN is supported by a special Recruitment Grant from the Monash University Faculty of Medicine Nursing and Health Sciences with funding from the State Government of Victoria and the Australian Government. We thank Bernd Bukau (ZMBH, Heidelberg University, Germany) and Harm H. Kampinga (Department of Biomedical Sciences of Cells & Systems, University of Groningen, The Netherlands) for their invaluable support and sharing of reagents, Holger Lorenz (ZMBH Imaging Facility, Heidelberg University, Germany) for his support with confocal microscopy and image processing, and Claire Hirst (ARMI, Monash University, Australia) for critical reading of the manuscript.

....

Materials

NameCompanyCatalog NumberComments
37% FormaldehydeMerck103999
AcetoneSigma-Aldrich32201
anti-DNAJA2 antibodyAbcamab157216
anti-DNAJB1 AntibodyEnzo Life SciencesADI-SPA-450
anti-DnaK antibodyIn house
anti-mCherry antibodyAbcamab125096
anti-Sis1 AntibodyCosmo Bio CorpCOP-080051
anti-Ydj1 antibodyStressMarq Biosciences SMC-150,
anti-YFP antibodyIn house
Coplin slide-staining jarSigma-AldrichS5516
Diagnostic slidesMarienfeld1216530
DMEMThermo-Fischer31966021
DuoLink In Situ Detection Reagents OrangeSigma-AldrichDUO92007
DuoLink In Situ Mounting Medium + DAPISigma-AldrichDUO82040
DuoLink In Situ PLA Probe Anti-Mouse MINUSSigma-AldrichDUO92004
DuoLink In Situ PLA Probe Anti-Rabbit PLUSSigma-AldrichDUO92002
DuoLink In Situ Wash Buffers, FluorescenceSigma-AldrichDUO82049
Fetal Calf SerumThermo-Fischer10082147
LysozymeSigma-Aldrich62971
MethanolSigma-Aldrich32213
ParaformaldehydeSigma-AldrichP6148
Penicillin/StreptomycinThermo-Fischer15070063
Poly-L-LysineSigma-AldrichP47-07
SorbitolSigma-AldrichS7547
Triton-X100Merck108643
TrypsinThermo-Fischer25300096
Tween-20Sigma-AldrichP1379
Zymolase 100T / / LyticaseUnited States BiologicalZ1004

References

  1. Soderberg, O., et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nature Methods. 3 (12), 995-1000 (2006).
  2. Weibrecht, I., et al. Proximity li....

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In Situ MonitoringTransient Protein InteractionsChaperone ComplexesProteostasisProkaryotic And Eukaryotic CellsMicrobial InfectionsProtein AssembliesImmunohistochemistryImmunofluorescenceELISAImmunoprecipitationsPoly L LysineParaformaldehydeTriton X100S Cerevisiae

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