Novel Role of ATPase Subunit C Targeting Peptides Beyond Mitochondrial Protein Import

In mammals, subunit c of the F1F0-ATP synthase has three isoforms (P1, P2, and P3). These isoforms differ by their cleavable mitochondrial targeting peptides, whereas the mature peptides are identical. To investigate this apparent genetic redundancy, we knocked down each of the three subunit c isoform by RNA interference in HeLa cells. Silencing any of the subunit c isoforms individually resulted in an ATP synthesis defect, indicating that these isoforms are not functionally redundant. We found that subunit c knockdown impaired the structure and function of the mitochondrial respiratory chain. In particular, P2 silencing caused defective cytochrome oxidase assembly and function. Because the expression of exogenous P1 or P2 was able to rescue the respective silencing phenotypes, but the two isoforms were unable to cross-complement, we hypothesized that their functional specificity resided in their targeting peptides. In fact, the expression of P1 and P2 targeting peptides fused to GFP variants rescued the ATP synthesis and respiratory chain defects in the silenced cells. Our results demonstrate that the subunit c isoforms are nonredundant, because they differ functionally by their targeting peptides, which, in addition to mediating mitochondrial protein import, play a yet undiscovered role in respiratory chain maintenance.

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Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes

Eukaryotic Cell ◽

10.1128/ec.00313-08 ◽

2008 ◽

Vol 8 (1) ◽

pp. 19-26 ◽

Cited By ~ 41

Author(s):

Ross F. Waller ◽

Carole Jabbour ◽

Nickie C. Chan ◽

Nermin Celik ◽

Vladimir A. Likić ◽

...

Keyword(s):

Outer Membrane ◽

Protein Import ◽

Markov Models ◽

Mitochondrial Protein ◽

Atp Synthesis ◽

Cellular Level ◽

Outer Membrane Receptor ◽

Mitochondrial Protein Import ◽

Severe Reduction ◽

Close Relatives

ABSTRACT Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.

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Structure and function of Tim14 and Tim16, the J and J-like components of the mitochondrial protein import motor

The EMBO Journal ◽

10.1038/sj.emboj.7601334 ◽

2006 ◽

Vol 25 (19) ◽

pp. 4675-4685 ◽

Cited By ~ 78

Author(s):

Dejana Mokranjac ◽

Gleb Bourenkov ◽

Kai Hell ◽

Walter Neupert ◽

Michael Groll

Keyword(s):

Protein Import ◽

Mitochondrial Protein ◽

Structure And Function ◽

Mitochondrial Protein Import ◽

And Function

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Preservation of Mitochondrial Structure and Function after Bid- or Bax-Mediated Cytochrome c Release

The Journal of Cell Biology ◽

10.1083/jcb.150.5.1027 ◽

2000 ◽

Vol 150 (5) ◽

pp. 1027-1036 ◽

Cited By ~ 173

Author(s):

Oliver von Ahsen ◽

Christian Renken ◽

Guy Perkins ◽

Ruth M. Kluck ◽

Ella Bossy-Wetzel ◽

...

Keyword(s):

Cytochrome C ◽

Protein Import ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Caspase Activation ◽

Cytochrome C Release ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

And Function

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1–mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

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Effect of denervation-induced muscle disuse on mitochondrial protein import

AJP Cell Physiology ◽

10.1152/ajpcell.00181.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. C138-C145 ◽

Cited By ~ 38

Author(s):

Kaustabh Singh ◽

David A. Hood

Keyword(s):

Protein Import ◽

Mitochondrial Protein ◽

Ros Generation ◽

Ros Production ◽

Mitochondrial Protein Import ◽

Mitochondrial Content ◽

Muscle Disuse ◽

The Matrix ◽

And Function ◽

Mitochondrial Heat Shock Protein

This study determined whether muscle disuse affects mitochondrial protein import and whether changes in protein import are related to mitochondrial content and function. Protein import was measured using a model of unilateral peroneal nerve denervation in rats for 3 ( n = 10), 7 ( n = 12), or 14 ( n = 14) days. We compared the import of preproteins into the matrix of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria isolated from the denervated and the contralateral control tibialis anterior muscles. Denervation led to 50% and 29% reductions in protein import after 14 days of disuse in SS and IMF mitochondria, respectively. This was accompanied by significant decreases in mitochondrial state 3 respiration, muscle mass, and whole muscle cytochrome c oxidase activity. To investigate the mechanisms involved, we assessed disuse-related changes in 1) protein import machinery components and 2) mitochondrial function, reflected by respiration and reactive oxygen species (ROS) production. Denervation significantly reduced the expression of translocases localized in the inner membrane (Tim23), outer membrane (Tom20), and mitochondrial heat shock protein 70 (mtHsp70), especially in the SS subfraction. Denervation also resulted in elevated ROS generation, and exogenous ROS was found to markedly reduce protein import. Thus our data indicate that protein import kinetics are closely related to alterations in mitochondrial respiratory capacity ( r = 0.95) and are negatively impacted by ROS. Deleterious changes in the protein import system likely facilitate the reduction in mitochondrial content and the increase in organelle dysfunction (i.e., increased ROS production and decreased respiration) during chronic disuse, which likely contribute to the activation of degradative pathways leading to muscle atrophy.

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3P-095 NMR analyses on the interactions between Tim21 for mitochondrial protein import and Qcr6 of the respiratory chain complex in mitochondria(The 46th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.48.s142_2 ◽

2008 ◽

Vol 48 (supplement) ◽

pp. S142

Author(s):

Takashi Yashiro ◽

Takashi Yokoyama ◽

Keiko Morishita ◽

Masahiro Maeda ◽

Takaki Momose ◽

...

Keyword(s):

Annual Meeting ◽

Respiratory Chain ◽

Protein Import ◽

Mitochondrial Protein ◽

Chain Complex ◽

Respiratory Chain Complex ◽

Mitochondrial Protein Import ◽

Biophysical Society

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Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration

Life ◽

10.3390/life11050432 ◽

2021 ◽

Vol 11 (5) ◽

pp. 432

Author(s):

Hope I. Needs ◽

Margherita Protasoni ◽

Jeremy M. Henley ◽

Julien Prudent ◽

Ian Collinson ◽

...

Keyword(s):

Protein Import ◽

Protein Translocation ◽

Mitochondrial Protein ◽

Nuclear Genome ◽

Mitochondrial Diseases ◽

Functional Protein ◽

Mitochondrial Protein Import ◽

Complex Assembly ◽

Respiratory Complex ◽

And Function

The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease.

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