Aim32 is a dual-localized 2Fe-2S mitochondrial protein that functions in redox quality control

Danyun Zhang,Owen R Dailey,Daniel J Simon,Kamilah Roca-Datzer,Yasaman Jami-Alahmadi,Mikayla S Hennen,James A Wohlschlegel,Carla M Koehler,Deepa V Dabir

doi:10.1016/j.jbc.2021.101135

Abstract

Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.

Highlights

Ixnsertion of disulfide bonds into substrate proteins [3,4,5,6,7]
A new partner protein, Altered inheritance of mitochondria 32 (Aim32), was enriched in the mitochondrial lysates from the erv1-12-HISPC yeast strain compared with the control, untagged WT strain (Fig. S1, and Table S2)
Because Aim32 was identified as an Erv1-binding partner, we investigated the redox status of Erv1 in mitochondria from WT and Δaim32 strains using thiol trapping with methyl-PEG-24-maleimide (Fig. 6C) [47]

Summary

Introduction

Ixnsertion of disulfide bonds into substrate proteins [3,4,5,6,7]. Substrates typically have a CX3C (i.e., the small Tim proteins) or CX9C motif (i.e., proteins such as Cox19 that may have a role in complex IV assembly and cross the membrane in a reduced and unfolded state). A new partner protein, Aim32, was enriched in the mitochondrial lysates from the erv1-12-HISPC yeast strain compared with the control, untagged WT strain (Fig. S1, and Table S2). Similar to cyt c and Mia40, a fraction (10%) of Aim32 copurified with Erv1; as a control, the tested proteins were not detected in the untagged WT strain (Fig. 1A).

Results

Conclusion