scholarly journals Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules

2018 ◽  
Vol 293 (43) ◽  
pp. 16899-16911 ◽  
Author(s):  
Leticia Veloso R. Franco ◽  
Chen-Hsien Su ◽  
Gavin P. McStay ◽  
George J. Yu ◽  
Alexander Tzagoloff
Keyword(s):  
Cytochrome Oxidase ◽  
Mitochondrial Gene ◽  
Membrane Insertion ◽  
Intermembrane Space ◽  
Gene Products ◽  
Binuclear Copper ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Copper Site ◽  
Oligomeric Complex

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11–13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450–550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

scholarly journals The Assembly Pathway of the Mitochondrial Carrier Translocase Involves Four Preprotein Translocases

2008 ◽  
Vol 28 (13) ◽  
pp. 4251-4260 ◽  
Author(s):  
Karina Wagner ◽  
Natalia Gebert ◽  
Bernard Guiard ◽  
Katrin Brandner ◽  
Kaye N. Truscott ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Important Principle ◽  
Highly Active ◽  
Mitochondrial Inner Membrane ◽  
Assembly Pathway ◽  
Oligomeric Complex ◽  
The Individual

ABSTRACT The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.

scholarly journals YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

2012 ◽  
Vol 23 (6) ◽  
pp. 1010-1023 ◽  
Author(s):  
Lukas Stiburek ◽  
Jana Cesnekova ◽  
Olga Kostkova ◽  
Daniela Fornuskova ◽  
Kamila Vinsova ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Membrane Protein ◽  
Respiratory Chain ◽  
Integral Membrane Protein ◽  
Intermembrane Space ◽  
Wild Type ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Human Orthologue ◽  
Excessive Accumulation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i‑AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600–1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

Inhibition by Oxytetracycline of the Development of the Enhanced Response to Noradrenaline in Cold-Acclimated Rats: A New Approach to the Study of Nonshivering Thermogenesis

1971 ◽  
Vol 49 (6) ◽  
pp. 545-553 ◽  
Author(s):  
Jean Himms–Hagen
Keyword(s):  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Cytochrome Oxidase ◽  
Oxidase Activity ◽  
Metabolic Response ◽  
Inner Membrane ◽  
Cytochrome Oxidase Activity ◽  
Mitochondrial Inner Membrane ◽  
Brown Adipose

The aim of these experiments was to depress the increased metabolic activity of the brown adipose tissue in the intact rat during acclimation to cold in order to elucidate further the possible thermogenic and endocrine functions of this tissue. The antibiotic oxytetracycline was administered twice daily for 2 weeks to rats living at 4 °C in an attempt to inhibit the proliferation of mitochondria and of mitochondrial inner membrane known to occur in the brown adipose tissue in response to cold; control rats received saline during the same period. Total cytochrome oxidase activity served as an index of the amount of mitochondrial inner membrane in brown adipose tissue, liver, and skeletal muscle. The development of an enhanced calorigenic response to intravenously infused noradrenaline served as an index of the extent of acclimation to cold.Treatment with oxytetracycline inhibited both the cold-induced increase in cytochrome oxidase activity in brown adipose tissue and the cold-induced development of an enhanced calorigenic response to noradrenaline in the intact rats; a direct correlation was noted between the amount of cytochrome oxidase in brown adipose tissue and the size of the metabolic response to noradrenaline of the intact animals. However, the amount of oxygen that could be consumed by the total cytochrome oxidase in the brown adipose tissue was itself too small to account for the increase in oxygen consumption by the rat. Treatment of the rats with oxytetracycline did not alter the cold-induced growth of brown adipose tissue (as judged by the increase in wet weight and the increase in total protein); it also did not alter the cytochrome oxidase activities of liver or skeletal muscle. The effect of oxytetracycline seems, therefore, to be fairly specific for the mitochondria of the most rapidly dividing tissue, the brown adipose tissue. The conclusion is drawn that a protein synthesized in the mitochondria of the brown adipose tissue in response to cold is essential for adaptation to cold.

scholarly journals Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

2009 ◽  
Vol 186 (6) ◽  
pp. 793-803 ◽  
Author(s):  
Rachel M. DeVay ◽  
Lenin Dominguez-Ramirez ◽  
Laura L. Lackner ◽  
Suzanne Hoppins ◽  
Henning Stahlberg ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Coiled Coil ◽  
Intermembrane Space ◽  
Dependent Manner ◽  
Related Protein ◽  
Mitochondrial Membranes ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
In Trans

Two dynamin-related protein (DRP) families are essential for fusion of the outer and inner mitochondrial membranes, Fzo1 (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals), respectively. Fzo1/Mfns possess two medial transmembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/Opa1 are present in cells as long (l) isoforms that are anchored via the N terminus to the inner membrane, and short (s) isoforms were predicted to be soluble in the intermembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in membrane fusion. Our analysis indicates that in the absence of a membrane, l- and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

scholarly journals Formation of Membrane-bound Ring Complexes by Prohibitins in Mitochondria

2005 ◽  
Vol 16 (1) ◽  
pp. 248-259 ◽  
Author(s):  
Takashi Tatsuta ◽  
Kirstin Model ◽  
Thomas Langer
Keyword(s):  
Cell Cycle Progression ◽  
Coiled Coil ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
High Molecular Weight Complex ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
Membrane Bound ◽  
Ring Complexes ◽  
Carboxy Terminal

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of ∼1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of ∼120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of ∼270 × 200 Å. Implications of these findings for proposed cellular activities of prohibitins are discussed.

scholarly journals Tim18p, a New Subunit of the TIM22 Complex That Mediates Insertion of Imported Proteins into the Yeast Mitochondrial Inner Membrane

2000 ◽  
Vol 20 (4) ◽  
pp. 1187-1193 ◽  
Author(s):  
Carla M. Koehler ◽  
Michael P. Murphy ◽  
Nikolaus A. Bally ◽  
Danielle Leuenberger ◽  
Wolfgang Oppliger ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Integral Membrane Protein ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Protein Insertion ◽  
Mitochondrial Inner Membrane ◽  
Precursor Proteins ◽  
Synthetically Lethal

ABSTRACT Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

scholarly journals The potential mechanism of mitochondrial dysfunction in septic cardiomyopathy

2018 ◽  
Vol 46 (6) ◽  
pp. 2157-2169 ◽  
Author(s):  
Pan Pan ◽  
Xiaoting Wang ◽  
Dawei Liu
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Gene ◽  
Mitochondrial Fission ◽  
Organ Damage ◽  
Septic Cardiomyopathy ◽  
Mitochondrial Uncoupling ◽  
Heart Research ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Proton Leakage

Septic cardiomyopathy is one of the most serious complications of sepsis or septic shock. Basic and clinical research has studied the mechanism of cardiac dysfunction for more than five decades. It has become clear that myocardial depression is not related to hypoperfusion. As the heart is highly dependent on abundant adenosine triphosphate (ATP) levels to maintain its contraction and diastolic function, impaired mitochondrial function is lethally detrimental to the heart. Research has shown that mitochondria play an important role in organ damage during sepsis. The mitochondria-related mechanisms in septic cardiomyopathy have been discussed in terms of restoring mitochondrial function. Mitochondrial uncoupling proteins located in the mitochondrial inner membrane can promote proton leakage across the mitochondrial inner membrane. Recent studies have demonstrated that proton leakage is the essential regulator of mitochondrial membrane potential and the generation of reactive oxygen species (ROS) and ATP. Other mechanisms involved in septic cardiomyopathy include mitochondrial ROS production and oxidative stress, mitochondria Ca2+ handling, mitochondrial DNA in sepsis, mitochondrial fission and fusion, mitochondrial biogenesis, mitochondrial gene regulation and mitochondria autophagy. This review will provide an overview of recent insights into the factors contributing to septic cardiomyopathy.

scholarly journals Phosphatidylserine transport by Ups2–Mdm35 in respiration-active mitochondria

2016 ◽  
Vol 214 (1) ◽  
pp. 77-88 ◽  
Author(s):  
Non Miyata ◽  
Yasunori Watanabe ◽  
Yasushi Tamura ◽  
Toshiya Endo ◽  
Osamu Kuge
Keyword(s):  
Yeast Cells ◽  
Intermembrane Space ◽  
Diauxic Shift ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Functions ◽  
Phospholipid Transfer ◽  
Metabolic Transition ◽  
Mitochondrial Intermembrane Space

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2–Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2–Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2–Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state.

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