In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, 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 microscopybased 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.

Replicative aging impedes stress-induced assembly of a key human protein disaggregase

“…PLA was performed as previously described. 14,26 . Cells were washed 2x with PBS, fixed with 4% paraformaldehyde in PBS, and the proximity ligation assay was performed according to the DUOLINK manufacturer’s guideline for mammalian cells (Sigma-Aldrich).…”

“…Next, we optimized and used the in situ proximity ligation assay (PLA) 14,26 to trace the assembly of trimeric Hsp70 disaggregases in human cells (Fig. 1a).…”

“…12 The Hsp110 cochaperone, which functions primarily as a nucleotide exchange factor (NEF) for Hsp70, was added to reactions to power protein disaggregation efficiency. 25 Next, we optimized and used the in situ proximity ligation assay (PLA) 14,26 to trace the assembly of trimeric Hsp70 disaggregases in human cells (Fig. 1a).…”