Effect of denervation-induced muscle disuse on mitochondrial protein import

2011 ◽  
Vol 300 (1) ◽  
pp. C138-C145 ◽  
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.

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

2010 ◽  
Vol 21 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Cristofol Vives-Bauza ◽  
Jordi Magrané ◽  
Antoni L. Andreu ◽  
Giovanni Manfredi
Keyword(s):  
Respiratory Chain ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Atp Synthesis ◽  
Mitochondrial Respiratory Chain ◽  
Genetic Redundancy ◽  
Mitochondrial Protein Import ◽  
Atpase Subunit ◽  
Subunit C ◽  
And Function

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.

scholarly journals Mitochondrial assembly: protein import

2004 ◽  
Vol 63 (2) ◽  
pp. 293-300 ◽  
Author(s):  
David A. Hood ◽  
Anna-Maria Joseph
Keyword(s):  
Molecular Chaperones ◽  
Mammalian Cells ◽  
Protein Import ◽  
Contractile Activity ◽  
Mitochondrial Protein ◽  
Energy Status ◽  
Mitochondrial Protein Import ◽  
Physiological Importance ◽  
The Matrix ◽  
Import Receptors

The protein import process of mitochondria is vital for the assembly of the hundreds of nuclear-derived proteins into an expanding organelle reticulum. Most of our knowledge of this complex multisubunit network comes from studies of yeast and fungal systems, with little information known about the protein import process in mammalian cells, particularly skeletal muscle. However, growing evidence indicates that the protein import machinery can respond to changes in the energy status of the cell. In particular, contractile activity, a powerful inducer of mitochondrial biogenesis, has been shown to alter the stoichiometry of the protein import apparatus via changes in several protein import machinery components. These adaptations include the induction of cytosolic molecular chaperones that transport precursors to the matrix, the up-regulation of outer membrane import receptors, and the increase in matrix chaperonins that facilitate the import and proper folding of the protein for subsequent compartmentation in the matrix or inner membrane. The physiological importance of these changes is an increased capacity for import into the organelle at any given precursor concentration. Defects in the protein import machinery components have been associated with mitochondrial disorders. Thus, contractile activity may serve as a possible mechanism for up-regulation of mitochondrial protein import and compensation for mitochondrial phenotype alterations observed in diseased muscle.

scholarly journals Tim23–Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import

2009 ◽  
Vol 184 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasushi Tamura ◽  
Yoshihiro Harada ◽  
Takuya Shiota ◽  
Koji Yamano ◽  
Kazuaki Watanabe ◽  
...  
Keyword(s):  
Motor Proteins ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
The Matrix ◽  
General Protein ◽  
Mitochondrial Hsp70

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.

Structural basis of mitochondrial protein import by the TIM complex

2021 ◽  
Author(s):  
Sue Im Sim ◽  
Yuanyuan Chen ◽  
Eunyong Park
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Structural Information ◽  
Mitochondrial Protein ◽  
Structural Basis ◽  
Mitochondrial Protein Import ◽  
Conducting Channel ◽  
The Core ◽  
The Matrix ◽  
Membrane Envelope

Mitochondria import nearly all their ~1,000-2,000 constituent proteins from the cytosol across their double membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM complex (also known as TIM23 complex), mediates import of presequence-containing proteins into the mitochondrial matrix and inner membrane. Among ~10 different subunits of the complex, the essential multi-pass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel. However, the mechanism of TIM-mediated protein import remains uncertain due to a lack of structural information on the complex. Here, we have determined the cryo-EM structure of the core TIM complex (Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. We show that, contrary to the prevailing model, Tim23 and Tim17 do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Remarkably, our data suggest that the cavity of Tim17 itself forms the protein translocation path whereas Tim23 plays a structural role. We also show how the Tim17-Tim23 heterodimer associates with the scaffold protein Tim44 and J-domain proteins to mediate Hsp70-driven polypeptide transport into the matrix. Our work provides the structural foundation to understand the mechanism of TIM-mediated protein import and sorting, a central pathway in mitochondrial biogenesis.

Contractile activity-induced adaptations in the mitochondrial protein import system

1998 ◽  
Vol 274 (5) ◽  
pp. C1380-C1387 ◽  
Author(s):  
Mark Takahashi ◽  
Alan Chesley ◽  
Damien Freyssenet ◽  
David A. Hood
Keyword(s):  
Outer Membrane ◽  
Malate Dehydrogenase ◽  
Mitochondrial Biogenesis ◽  
Protein Import ◽  
Contractile Activity ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
The Matrix ◽  
Stimulating Factor ◽  
Import Receptor

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4- to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

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

2006 ◽  
Vol 25 (19) ◽  
pp. 4675-4685 ◽  
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

scholarly journals Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

1998 ◽  
Vol 9 (9) ◽  
pp. 2577-2593 ◽  
Author(s):  
Alison J. Davis ◽  
Kathleen R. Ryan ◽  
Robert E. Jensen
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Amino Terminal ◽  
Transmembrane Segments ◽  
Charged Amino Acids ◽  
The Matrix ◽  
Positively Charged

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.

scholarly journals A chimeric mitochondrial precursor protein with internal disulfide bridges blocks import of authentic precursors into mitochondria and allows quantitation of import sites.

1988 ◽  
Vol 107 (6) ◽  
pp. 2037-2043 ◽  
Author(s):  
D Vestweber ◽  
G Schatz
Keyword(s):  
Trypsin Inhibitor ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Precursor Protein ◽  
Disulfide Bridges ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Inner Membrane ◽  
Precursor Proteins ◽  
The Matrix ◽  
Pancreatic Trypsin Inhibitor

Bovine pancreatic trypsin inhibitor (which contains three intramolecular disulfide bridges) was chemically coupled to the COOH terminus of a purified artificial mitochondrial precursor protein. When the resulting chimeric precursor was presented to energized isolated yeast mitochondria, its trypsin inhibitor moiety prevented the protein from completely entering the organelle; the protein remained stuck across both mitochondrial membranes, with its NH2 terminus in the matrix and its trypsin inhibitor moiety still exposed on the mitochondrial surface. The incompletely imported protein appeared to "jam" mitochondrial protein import sites since it blocked import of three authentic mitochondrial precursor proteins; it did not collapse the potential across the mitochondrial inner membrane. Quantification of the inhibition indicated that each isolated mitochondrial particle contains between 10(2) and 10(3) protein import sites.

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

2000 ◽  
Vol 150 (5) ◽  
pp. 1027-1036 ◽  
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.

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

Life ◽  
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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