The interaction of the mitochondrial protein importer TOMM34 with HSP70 is regulated by TOMM34 phosphorylation and binding to 14-3-3 adaptors

Filip Trcka,Michal Durech,Pavla Vankova,Veronika Vandova,Oliver Simoncik,Daniel Kavan,Borivoj Vojtesek,Petr Muller,Petr Man

doi:10.1074/jbc.ra120.012624

Abstract

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

Highlights

Two highly conserved families of molecular chaperones, heat shock protein 70 (HSP70) and HSP90, represent core machineries surveying folding and conformational status of cellular proteome [1, 2]
Translocase of outer mitochondrial membrane 34 (TOMM34)·HSP70 complex formation requires both accommodation of the HSP70 EEVD C-terminal motif by the TOMM34 tetratricopeptide repeat 1 (TPR1) domain and docking of so-far-unknown determinants in the TOMM34 interdomain linker to HSP70 in the ATP-bound conformation [21, 23]
We have shown that Ser93 (TPR1 domain) and Ser160 TOMM34 residues are effectively phosphorylated by protein kinase A (PKA) (Fig. 1 and Tables S2 and S3)

Summary

Introduction

Two highly conserved families of molecular chaperones, HSP70 and HSP90, represent core machineries surveying folding and conformational status of cellular proteome [1, 2]. TOMM34 phosphorylation induces structural changes in its TPR domains that are augmented by 14-3-3 protein binding and modulate the interaction of TOMM34 with HSP70.

Results

Conclusion