scholarly journals A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

2007 ◽  
Vol 179 (7) ◽  
pp. 1355-1363 ◽  
Author(s):  
Hidenori Otera ◽  
Yohsuke Taira ◽  
Chika Horie ◽  
Yurina Suzuki ◽  
Hiroyuki Suzuki ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Benzodiazepine Receptor ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Transmembrane Segments ◽  
Import Receptors ◽  
Mitochondrial Intermembrane Space

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.

scholarly journals Mitochondrial inner-membrane protease Yme1 degrades outer-membrane proteins Tom22 and Om45

2017 ◽  
Vol 217 (1) ◽  
pp. 139-149 ◽  
Author(s):  
Xi Wu ◽  
Lanlan Li ◽  
Hui Jiang
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Ubiquitin Proteasome System ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Mitochondrial Proteome ◽  
Mitochondrial Inner Membrane ◽  
Ubiquitin Proteasome

Mitochondria are double-membraned organelles playing essential metabolic and signaling functions. The mitochondrial proteome is under surveillance by two proteolysis systems: the ubiquitin–proteasome system degrades mitochondrial outer-membrane (MOM) proteins, and the AAA proteases maintain the proteostasis of intramitochondrial compartments. We previously identified a Doa1–Cdc48-Ufd1-Npl4 complex that retrogradely translocates ubiquitinated MOM proteins to the cytoplasm for degradation. In this study, we report the unexpected identification of MOM proteins whose degradation requires the Yme1-Mgr1-Mgr3 i-AAA protease complex in mitochondrial inner membrane. Through immunoprecipitation and in vivo site-specific photo–cross-linking experiments, we show that both Yme1 adapters Mgr1 and Mgr3 recognize the intermembrane space (IMS) domains of the MOM substrates and facilitate their recruitment to Yme1 for proteolysis. We also provide evidence that the cytoplasmic domain of substrate can be dislocated into IMS by the ATPase activity of Yme1. Our findings indicate a proteolysis pathway monitoring MOM proteins from the IMS side and suggest that the MOM proteome is surveilled by mitochondrial and cytoplasmic quality control machineries in parallel.

scholarly journals The Tom40 assembly process probed using the attachment of different intramitochondrial sorting signals

2012 ◽  
Vol 23 (20) ◽  
pp. 3936-3947 ◽  
Author(s):  
Takuya Shiota ◽  
Miyuki Maruyama ◽  
Mami Miura ◽  
Yasushi Tamura ◽  
Koji Yamano ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Assembly Process ◽  
Conducting Channel ◽  
Inner Membrane ◽  
Sorting Signals ◽  
Insight Into

The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a β-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effective in substituting for the requirement for small Tim proteins in the IMS for the translocation of Tom40 across the outer membrane. These results provide insight into the mechanism responsible for the precise delivery of β-barrel proteins to the outer mitochondrial membrane.

scholarly journals Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

2009 ◽  
Vol 20 (8) ◽  
pp. 2276-2285 ◽  
Author(s):  
Blanca Schafer ◽  
Joel Quispe ◽  
Vineet Choudhary ◽  
Jerry E. Chipuk ◽  
Teddy G. Ajero ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pore Formation ◽  
Outer Membrane Proteins ◽  
Mitochondrial Outer Membrane ◽  
Outer Membranes ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
In Vitro Systems ◽  
Large Round ◽  
Key Features

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.

scholarly journals Evolution of mitochondrial protein import – lessons from trypanosomes

2020 ◽  
Vol 401 (6-7) ◽  
pp. 663-676 ◽  
Author(s):  
André Schneider
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Protein Import ◽  
Outer Membrane Proteins ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
System A ◽  
Import Receptors ◽  
Inner Membrane Protein

AbstractThe evolution of mitochondrial protein import and the systems that mediate it marks the boundary between the endosymbiotic ancestor of mitochondria and a true organelle that is under the control of the nucleus. Protein import has been studied in great detail in Saccharomyces cerevisiae. More recently, it has also been extensively investigated in the parasitic protozoan Trypanosoma brucei, making it arguably the second best studied system. A comparative analysis of the protein import complexes of yeast and trypanosomes is provided. Together with data from other systems, this allows to reconstruct the ancestral features of import complexes that were present in the last eukaryotic common ancestor (LECA) and to identify which subunits were added later in evolution. How these data can be translated into plausible scenarios is discussed, providing insights into the evolution of (i) outer membrane protein import receptors, (ii) proteins involved in biogenesis of α-helically anchored outer membrane proteins, and (iii) of the intermembrane space import and assembly system. Finally, it is shown that the unusual presequence-associated import motor of trypanosomes suggests a scenario of how the two ancestral inner membrane protein translocases present in LECA evolved into the single bifunctional one found in extant trypanosomes.

scholarly journals Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.

1991 ◽  
Vol 174 (5) ◽  
pp. 1167-1177 ◽  
Author(s):  
J Vuopio-Varkila ◽  
G K Schoolnik
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
De Novo ◽  
Outer Membrane Proteins ◽  
Temporal Correlation ◽  
Enteropathogenic Escherichia Coli ◽  
E Coli

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

scholarly journals The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

2011 ◽  
Vol 194 (3) ◽  
pp. 387-395 ◽  
Author(s):  
Thomas Becker ◽  
Lena-Sophie Wenz ◽  
Vivien Krüger ◽  
Waltraut Lehmann ◽  
Judith M. Müller ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Mitochondrial Outer Membrane ◽  
Limited Information ◽  
Mitochondrial Import ◽  
Transmembrane Segments ◽  
Membrane Complex ◽  
Complex Functions ◽  
Precursor Proteins

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.

scholarly journals Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

2011 ◽  
Vol 63 (2) ◽  
pp. 174-182 ◽  
Author(s):  
Bruno D'Alessandro ◽  
Leticia M. S. Lery ◽  
Wanda M. A. Krüger ◽  
Analía Lima ◽  
Claudia Piccini ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Differential Expression ◽  
Proteus Mirabilis ◽  
Proteomic Analysis ◽  
Outer Membrane Proteins

scholarly journals Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization

2020 ◽  
Vol 295 (6) ◽  
pp. 1623-1636 ◽  
Author(s):  
Tomomi Kuwana ◽  
Louise E. King ◽  
Katia Cosentino ◽  
Julian Suess ◽  
Ana J. Garcia-Saez ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Permeabilization ◽  
Size Exclusion ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Double Knockout ◽  
Apoptosis Pathway ◽  
Bax Protein ◽  
Intrinsic Apoptosis Pathway

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2–associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

scholarly journals An iron-regulated outer-membrane protein of Proteus mirabilis is a haem receptor that plays an important role in urinary tract infection and in in vivo growth

2007 ◽  
Vol 56 (12) ◽  
pp. 1600-1607 ◽  
Author(s):  
Analía Lima ◽  
Pablo Zunino ◽  
Bruno D'Alessandro ◽  
Claudia Piccini
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Outer Membrane ◽  
Proteus Mirabilis ◽  
Outer Membrane Proteins ◽  
Mammalian Host ◽  
Tract Infections

Proteus mirabilis, a common cause of urinary tract infections, expresses iron-regulated outer-membrane proteins (OMPs) in response to iron restriction. It has been suggested that a 64 kDa OMP is involved in haemoprotein uptake and that this might have a role in pathogenesis. In order to confirm this hypothesis, this study generated a P. mirabilis mutant strain (P7) that did not express the 64 kDa OMP, by insertion of the TnphoA transposon. The nucleotide sequence of the interrupted gene revealed that it corresponded to a haemin receptor precursor. Moreover, in vitro growth assays showed that the mutant was unable to grow using haemoglobin and haemin as unique iron sources. The authors also carried out in vivo growth and infectivity assays and demonstrated that P7 was not able to survive in an in vivo model and was less efficient than wild-type strain Pr 6515 in colonizing the urinary tract. These results confirmed that the P. mirabilis 64 kDa iron-regulated OMP is a haem receptor that has an important role for survival and multiplication of these bacteria in the mammalian host and in the development of urinary tract infection.

scholarly journals The Dynamin-Related Gtpase, Mgm1p, Is an Intermembrane Space Protein Required for Maintenance of Fusion Competent Mitochondria

2000 ◽  
Vol 151 (2) ◽  
pp. 341-352 ◽  
Author(s):  
Edith D. Wong ◽  
Jennifer A. Wagner ◽  
Steven W. Gorsich ◽  
J. Michael McCaffery ◽  
Janet M. Shaw ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Fission ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Intermembrane Space ◽  
Direct Role ◽  
Membrane Fission ◽  
Mitochondrial Intermembrane Space

Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.

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