Phosphorylation Enhances Mitochondrial Targeting of GSTA4-4 through Increased Affinity for Binding to Cytoplasmic Hsp70

Marie-Anne Robin,Subbuswamy K Prabu,Haider Raza,Hindupur K Anandatheerthavarada,Narayan G Avadhani

doi:10.1074/jbc.m301807200

Marie-Anne Robin, Subbuswamy K Prabu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m301807200

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Abstract

Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.

Highlights

GSTA4-41 belongs to a major group of enzymes involved in detoxification and protection of cells against chemical and oxidative stress
We have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA
Our results show that the N-terminal deletion had no effect on the mitochondrial targeting of the protein, whereas the C-terminal deletions inhibited it in vivo (Fig. 5B) as well as in vitro (Fig. 5C) transport, indicating that the targeting signal might reside at the C terminus of GSTA4-4

Summary

Introduction

GSTA4-41 belongs to a major group of enzymes involved in detoxification and protection of cells against chemical and oxidative stress. The use of translation mix containing PKA or PKC in the in vitro import experiments in Fig. 3A above leaves open the possibility that phosphorylation of mitochondrial membrane proteins contribute to the increased import.

Results

Conclusion