MGARP is ultrastructurally located in the inner faces of mitochondrial membranes

https://doi.org/10.1016/j.bbrc.2019.06.028Get rights and content

Highlights

  • More MGARPs localize in the mitochondrial inner/cristae membranes than in the outer membranes.

  • MGARP overexpression causes both mitochondrial remodeling and cristae-shaping.

  • Cristae are the major platforms for MGARP functioning.

  • MGARP without its transmembrane domain loses its effect on mitochondrial architecture.

Abstract

Mitochondria, the centers of energy production, are highly organized with inner membranes, cristae and outer membranes. The mitochondrial architecture determines their functions in all cellular processes. Changes in the mitochondrial ultrastructure are tightly related to a wide variety of diseases. MGARP, a mitochondria-localized protein, was predicted by bioinformatics and confirmed by cellular and biochemical methods to be located in mitochondria, but there is no direct and clear evidence for its precise location. This report demonstrates the precise ultrastructural location of MGARP within mitochondria by the ascorbate peroxidase 2 (APEX2) system in combination with electron microscopy (EM). EM revealed that more MGARP is located in the inner/cristae membranes, with its C-terminus at the inner faces of the intramembrane spaces, than in the outer membranes. MGARP overexpression caused both mitochondrial remodeling and cristae shaping, leading to the collapse of the mitochondrial network. The mitochondrial morphologies in MGARP-overexpressing cells were diverse; the cells became round or short, and their cristae were deformed and became discontinuous or circular. An engineered MGARP mutant deficient in its transmembrane domain no longer localized to the mitochondria and lost its effects on mitochondrial structure, confirming that the localization of MGARP in the mitochondria depends on its structural integrity. Collectively, our findings define the location of MGARP within the mitochondria, which is associated with its functional implications for the architecture and organization of mitochondria.

Introduction

Mitochondria play important roles in various physiological processes, including energy production, metabolism and autophagy [1]. Mitochondrial dynamics and quality control are tightly associated with sickness and health. Defects in mitochondria caused by mutations in mitochondrial and/or nuclear DNA lead to diverse and complex human diseases [2]. Within cells, mitochondria form a dynamic, interconnected network that is intimately integrated with other cellular compartments [3]. A mitochondrion consists of an outer membrane, an intermembrane space, an inner membrane, cristae and a matrix. Different regions of mitochondria carry out specialized functions. At the outer membrane, proteins, nucleotides, ions, fatty acids and metabolites can be transported between the cytosol and the intermembrane space [4,5]. At the inner mitochondrial membrane (IMM)/cristae, energy conversion and ATP production occur, and proteins are shuttled into the matrix [[6], [7], [8]]. Mitochondrial cristae shape determines respiratory chain supercomplex assembly and respiratory efficiency [9]. Mitochondrial morphology and inner structure critically depend on mitochondria-shaping proteins [10].

Many proteins are located in the mitochondrial membranes. These mitochondrial proteins, many of which have important functions, usually contain a mitochondrial targeting signal [7,11,12]. Among mitochondria-localized proteins, the majority are encoded by nuclear DNA [13]. MGARP, a mitochondria-localized, glutamic acid-rich protein, is also encoded by a nuclear chromosome and was first discovered from a large-scale screen of the ovary [14]. MGARP was later demonstrated to be highly expressed in steroid tissues (ovary, testes and adrenal gland) and visual system [[15], [16], [17]]. MGARP is involved in steroidogenesis and negatively mediates neocortical development by regulating mitochondrial distribution and motility in neocortical neurons [15,18]. Its expression regulated by the hypothalamic–pituitary–gonadal axis (also called the HPG axis) and fluctuates with the estrogen level [17]. MGARP upregulation in U17 snoRNA-deficient cells promoted the formation of ER-mitochondrial contacts to regulate cholesterol flux to mitochondria [19].

However, there is no direct and clear evidence for the precise location of MGARP. A previous analysis of MGARP topology predicted that the C-terminus of MGARP is located inside the membrane, suggesting that the transmembrane domain (TMD) is in the outer mitochondrial membrane (OMM) with the C-terminus in the intermembrane space (IMS) [20]. Experiments performed by Li et al. demonstrated that osmotic shock alone and without trypsin did not alter MGARP protein abundance [21]. Combined treatment with osmotic shock and trypsin degraded MGARP, which strongly suggests that the C-terminus of MGARP is in the IMS, but MGARP was not released from the IMS following treatment with osmotic shock [21]. The precise localization of MGARP within mitochondria will help to clarify and understand these observations.

In this study, we established an enhanced ascorbate peroxidase 2 (APEX2) system [22] and combined this system with electron microscopy (EM) to monitor the precise location of MGARP within mitochondria. We found that MGARP proteins were predominantly located in the inner/cristae membranes, with less MGARP in the outer membranes, and that the C-terminus of MGARP was located in the inner faces of intermembrane spaces. In addition, MGARP overexpression (OE) caused mitochondria remodeling and cristae shaping. The transmembrane domain and the rest of the N-terminal regions of MGARP were essential for MGARP localization and function. Together, our studies show for the first time the precise location of MGARP and identify MGARP as a cristae-shaping protein.

Section snippets

Antibodies and reagents

Restriction and modifying enzymes were obtained from Takara Biotechnology (Dalian, China). Anti-myc and anti-GAPDH were purchased from ABclonal (Woburn, MA). Anti-V5 was purchased from HuaXingBio (Beijing, China). The fluorescent secondary antibodies TRITC-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse IgG and anti-mouse IgG were purchased from ZSGB-BIO (Beijing, China). The MGARP antibody was generated as previously described [17]. Hoechst 33342 was purchased from Sigma (St.

Results and discussion

Validation of the activity and localization of the APEX2 and MGARP + APEX2 fusion constructs by western blotting, immunostaining and light microscopy.

MGARP has a single transmembrane domain [[14], [15], [16],21], but there is no direct and clear evidence of its precise location within mitochondria. Thus, we established an enhanced ascorbate peroxidase 2 (APEX2) system in HeLa cells that can emit a robust signal through APEX2 activity that can be readily captured by electron microscopy (EM) [22,

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgements

It is grateful to Prof. Shaoyong Chen (Beth Israel Deaconess Medical Center, Harvard Medical School, USA) for carefully reading our manuscript and providing constructive suggestions. We sincerely thank Prof. Peng Zou (School of Chemistry and Molecular Engineering, Peking University), Prof. Li Yu, Prof. Jianbin Wang and Dr. Ying Li (School of Life Sciences, Tsinghua University) for help with the APEX system.

References (33)

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This paper is written to show appreciation for the work done by a graduate student, Tingyue Yan.

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