TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells.

Yuki Kawaguchi,Ikuo Shoji,Masato Taoka,Takamasa Uekita,Naomi Hachiya,Tohru Ichimura,Takahiro Takekiyo,Michael Sherman

doi:10.1371/journal.pone.0169436

Abstract

The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions.

Highlights

Protein aggregation occurs spontaneously through a decrease of free energy [1], this reaction is typically prevented in cells by the protein quality control machinery (QC)
That many other targets were more abundant in identified peptide number and sequence coverage than 14-3-3s under these conditions, including the following QC components: the chaperone HSP70 (HSPA1A) and its family members, several subunits of the chaperonin TRiC, and polyubiquitin B
HSP70 is a molecular chaperone that is central to the QC system, so we further analyzed this interaction through western blots with antibodies specific to ectopic myc-HSPA1A or endogenous HSP70

Summary

Introduction

Protein aggregation occurs spontaneously through a decrease of free energy [1], this reaction is typically prevented in cells by the protein quality control machinery (QC). We transiently transfected HEK293 cells (2–4 × 106) with FLAG-TRIM32 expression plasmids that were either with or without myc-HSP70 or K71S (5μg each).

Results

Conclusion