Elsevier

Mitochondrion

Volume 20, January 2015, Pages 43-51
Mitochondrion

MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism

https://doi.org/10.1016/j.mito.2014.10.003Get rights and content

Highlights

  • New regulator of mitochondrial metabolism

  • Acts in two cellular compartments, mitochondria and nucleus

  • Responds to oxidative or hypoxic stress

  • Imported to the mitochondrial intermembrane space by a Mia40-mediated pathway

Abstract

Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil–helix–coiled-coil–helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.

Introduction

Although signaling from the nucleus is primarily responsible for cell growth and development, including mitochondrial function (anterograde regulation), it has been clear for a number of years that mitochondrial signaling to the nucleus and the resultant response (retrograde regulation) is a widely used and conserved mechanism (Jazwinski, 2013, Liu and Butow, 2006). In yeast, retrograde regulation appears primarily to allow cells to adjust their metabolism to altered external conditions (Liu and Butow, 2006) whereas in mammalian cells more heterogeneous signals of mitochondrial dysfunction appear able to activate a nuclear response (Butow and Avadhani, 2004, Hansen et al., 2006). In both cases, but more so for mammalian cells, the nature of the pathways and the signals that lead to their activation are not well known. Retrograde regulation encompasses a wide variety of activities, including nutrient sensing, growth control, stress response, and aging. Here we focus on signaling involving stress of the cell's critical energy producing machinery.

Oxidative phosphorylation (OxPhos) within the mitochondria plays a pivotal role in cellular physiology by generating energy in the form of ATP by ATP-synthase via utilization of the mitochondrial membrane potential generated by the electron transport chain (ETC) (Hüttemann et al., 2012a). Maintaining appropriate energy balance in the face of environmental changes is a key regulatory challenge for cells, and a number of pathways have come to light. A well known one is the HIF-1 pathway that adapts cells to altered oxygen environment and its stresses (Semenza, 2012). Other pathways, such as the AMP kinase pathway, respond to changes in energy via adenine nucleotide levels (Mihaylova and Shaw, 2011) and the sirtuin pathway via changes in NADH/NAD+ levels (White and Schenk, 2012).

A recently identified regulator of mitochondrial function, MNRR1 (Mitochondria Nuclear Retrograde Regulator 1) (previously called coiled coil–helix–coiled coil–helix domain containing protein 2 (CHCHD2)) (Aras et al., 2013, Baughman et al., 2009), belongs to a class of eukaryotic twin CX9C proteins that are typified by the presence of two pairs of cysteine residues forming disulfide linkages that define a CHCH fold (Cavallaro, 2010, Koehler and Tienson, 2009). Each cysteine in the pair is separated by nine amino acids. These proteins are frequently localized to the mitochondrial intermembrane space (IMS). MNRR1 was first identified in a computational screen as a protein necessary for OxPhos (Baughman et al., 2009). Knockdown of MNRR1 in immortalized human fibroblasts reduced oxygen consumption rate in live cells by about 40%. Other functions have been ascribed to MNRR1 in mammalian cells and in model organisms. These include cell migration (Seo et al., 2010), resistance to the cytotoxic drug hemiasterlin in Caenorhabditis elegans (har-1 is the ortholog of human MNRR1) (Zubovych et al., 2010), and oxygen consumption in Saccharomyces cerevisiae (Levy et al., 2014) (Mic17 is a yeast MNRR1 homolog).

We have previously shown MNRR1 to function as a transcriptional activator for COX subunit 4 isoform 2 (COX4I2), with an enhanced response under low oxygen tension (Aras et al., 2013). Yeast one-hybrid selection and DNA binding assays confirmed that MNRR1 specifically binds a 13-bp highly conserved element in the COX4I2 promoter that we have termed the oxygen responsive element (ORE). Further, MNRR1 expression in these cells was stimulated by hypoxia. Knockdown of MNRR1 in primary rat pulmonary artery smooth muscle cells (PASMCs) decreased the expression of COX4I2 under hypoxic conditions. Notably, the transactivator function of MNRR1 at the COX4I2 promoter was independent of HIF-1α.

In the present study, we have characterized MNRR1 protein function. Our results indicate that MNRR1 is a bi-organellar protein predominantly localized to the mitochondria but with a fraction also present in the nucleus. In the mitochondria, MNRR1 binds to COX and is necessary for its optimal function in respiration. MNRR1 also plays a pivotal role in regulation of cellular ROS generation and oxidative stress such that cells with decreased MNRR1 display a distinct phenotype of mitochondrial dysfunction. In the nucleus MNRR1 stimulates transcription of a gene set that includes COX4I2 and itself.

Section snippets

MNRR1 binds to and regulates cytochrome c oxidase activity

To evaluate the role of MNRR1 in the functioning of COX, lysates of 293 cells that are wild-type and those in which MNRR1 expression is suppressed (293-MNRR1-KD) or enhanced (293-MNRR1-OE) were analyzed for COX activity. COX activity was responsive to manipulation of MNRR1 level, being about 50% higher in 293-MNRR1-OE cells and lower in the 293-MNRR1-KD cells compared to control cells (Fig. 1A). Respiration in knockdown cells was almost at baseline even in the presence of saturating

Discussion

We have shown that the twin CX9C protein MNRR1, here renamed from CHCHD2 to reflect functionality as MNRR1 (Mitochondria Nuclear Retrograde Regulator 1), is a regulator of mitochondrial metabolism that is active in two cellular organelles. In one organelle, the mitochondria, it binds to and regulates the activity of COX and in the nucleus it functions in trans to stimulate transcription of a subset of genes that includes COX4I2 and itself.

Surprisingly, MNRR1 binds directly to COX and is

Cell lines

Human embryonic kidney 293 cells (293 cells) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). A stable knockdown of MNRR1 was generated by transfecting 293 cells with a MNRR1 shRNA plasmid (Santa Cruz Biotechnology Inc., Dallas, TX, USA), followed by selection of puromycin resistant clones (293-MNRR1-KD). Cells overexpressing MNRR1 (293-MNRR1-OE) were generated by transfecting 293 cells with the MNRR1

Author contributions

SA, MB, IL and RS performed the experiments. SA, MH and LIG analyzed the results and participated in experimental design. SA, MH and LIG wrote the manuscript.

Acknowledgments

We thank Dr. Lobelia Samavati, Wayne State University, for providing us with the HA-Akt expression plasmid and Dr. Carla Koehler, UCLA, for MitoBloCK-6. The authors acknowledge Malati Vadapalli, Wayne State University, for the recombinant in vitro COX activity assay and Christopher Sinkler for assistance in microscopy.

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