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 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 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.

scholarly journals Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

1986 ◽  
Vol 6 (2) ◽  
pp. 626-634 ◽  
Author(s):  
G S Adrian ◽  
M T McCammon ◽  
D L Montgomery ◽  
M G Douglas
Keyword(s):  
Transmembrane Protein ◽  
Nuclear Gene ◽  
Translocator Protein ◽  
Gene Encoding ◽  
Inner Membrane ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
Membrane Anchoring ◽  
High Degree

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.

scholarly journals Dominant Negative Alleles ofSEC10Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

1998 ◽  
Vol 9 (7) ◽  
pp. 1725-1739 ◽  
Author(s):  
Dagmar Roth ◽  
Wei Guo ◽  
Peter Novick
Keyword(s):  
Plasma Membrane ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Dominant Negative ◽  
Secretory Vesicles ◽  
Cycle Progression ◽  
Vesicle Docking ◽  
Amino Terminal ◽  
Exocyst Complex ◽  
Carboxy Terminal

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeastSaccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

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 Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

1999 ◽  
Vol 10 (7) ◽  
pp. 2461-2474 ◽  
Author(s):  
Martin Kurz ◽  
Heiko Martin ◽  
Joachim Rassow ◽  
Nikolaus Pfanner ◽  
Michael T. Ryan
Keyword(s):  
Crossing Over ◽  
Intermembrane Space ◽  
Membrane Translocation ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Surface Receptors ◽  
Amino Terminal ◽  
Membrane Carriers ◽  
Positively Charged ◽  
Tim Proteins

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23–Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22–Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23–Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

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.

scholarly journals Cox18p Is Required for Export of the Mitochondrially EncodedSaccharomyces cerevisiaeCox2p C-Tail and Interacts with Pnt1p and Mss2p in the Inner Membrane

2002 ◽  
Vol 13 (4) ◽  
pp. 1122-1131 ◽  
Author(s):  
Scott A. Saracco ◽  
Thomas D. Fox
Keyword(s):  
Intermembrane Space ◽  
Biosynthetic Enzyme ◽  
Epitope Tag ◽  
Inner Membrane ◽  
Multiple Alleles ◽  
Functional Interactions ◽  
C Terminus ◽  
The Matrix ◽  
Inner Membrane Protein ◽  
Carboxy Terminal

The amino- and carboxy-terminal domains of mitochondrially encoded cytochrome c oxidase subunit II (Cox2p) are translocated out of the matrix to the intermembrane space. We have carried out a genetic screen to identify components required to export the biosynthetic enzyme Arg8p, tethered to the Cox2p C terminus by a translational gene fusion inserted into mtDNA. We obtained multiple alleles of COX18, PNT1, and MSS2, as well as mutations in CBP1 and PET309. Focusing on Cox18p, we found that its activity is required to export the C-tail of Cox2p bearing a short C-terminal epitope tag. This is not a consequence of reduced membrane potential due to loss of cytochrome oxidase activity because Cox2p C-tail export was not blocked in mitochondria lacking Cox4p. Cox18p is not required to export the Cox2p N-tail, indicating that these two domains of Cox2p are translocated by genetically distinct mechanisms. Cox18p is a mitochondrial integral inner membrane protein. The inner membrane proteins Mss2p and Pnt1p both coimmunoprecipitate with Cox18p, suggesting that they work together in translocation of Cox2p domains, an inference supported by functional interactions among the three genes.

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