The N-terminal 21 amino acids of a 70 kDa protein of the yeast mitochondrial outer membrane direct E. coli
 β-galactosidase into the mitochondrial matrix space in yeast cells

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1

The Journal of Cell Biology ◽

10.1083/jcb.200512079 ◽

2006 ◽

Vol 173 (5) ◽

pp. 645-650 ◽

Cited By ~ 90

Author(s):

Mafalda Escobar-Henriques ◽

Benedikt Westermann ◽

Thomas Langer

Keyword(s):

Outer Membrane ◽

Outer Membrane Proteins ◽

Critical Role ◽

Mitochondrial Fusion ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Central Component ◽

Mitochondrial Morphology ◽

Proteolytic Pathway ◽

F Box Protein

Mitochondrial morphology depends on balanced fusion and fission events. A central component of the mitochondrial fusion apparatus is the conserved GTPase Fzo1 in the outer membrane of mitochondria. Mdm30, an F-box protein required for mitochondrial fusion in vegetatively growing cells, affects the cellular Fzo1 concentration in an unknown manner. We demonstrate that mitochondrial fusion requires a tight control of Fzo1 levels, which is ensured by Fzo1 turnover. Mdm30 binds to Fzo1 and, dependent on its F-box, mediates proteolysis of Fzo1. Unexpectedly, degradation occurs along a novel proteolytic pathway not involving ubiquitylation, Skp1–Cdc53–F-box (SCF) E3 ubiquitin ligase complexes, or 26S proteasomes, indicating a novel function of an F-box protein. This contrasts to the ubiquitin- and proteasome-dependent turnover of Fzo1 in α-factor–arrested yeast cells. Our results therefore reveal not only a critical role of Fzo1 degradation for mitochondrial fusion in vegetatively growing cells but also the existence of two distinct proteolytic pathways for the turnover of mitochondrial outer membrane proteins.

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The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3382 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3382-3389 ◽

Cited By ~ 96

Author(s):

A Hönlinger ◽

M Kübrich ◽

M Moczko ◽

F Gärtner ◽

L Mallet ◽

...

Keyword(s):

Outer Membrane ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Receptor Complex ◽

Direct Role ◽

Double Deletion ◽

Fermentative Growth ◽

Limited Function ◽

Import Receptors ◽

In Transit

A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.

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Grx5 Is a Mitochondrial Glutaredoxin Required for the Activity of Iron/Sulfur Enzymes

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0517 ◽

2002 ◽

Vol 13 (4) ◽

pp. 1109-1121 ◽

Cited By ~ 325

Author(s):

Marı́a Teresa Rodrı́guez-Manzaneque ◽

Jordi Tamarit ◽

Gemma Bellı́ ◽

Joaquim Ros ◽

Enrique Herrero

Keyword(s):

Amino Acids ◽

Oxidative Damage ◽

Yeast Cells ◽

Mitochondrial Matrix ◽

Translation Product ◽

Iron Sulfur Clusters ◽

Growth Defects ◽

Iron Sulfur ◽

Reduction Of Iron ◽

Null Mutants

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression ofSSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.

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Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells

Journal of Virology ◽

10.1128/jvi.76.20.10485-10496.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10485-10496 ◽

Cited By ~ 59

Author(s):

Frédérique Weber-Lotfi ◽

André Dietrich ◽

Marcello Russo ◽

Luisa Rubino

Keyword(s):

Outer Membrane ◽

Fluorescent Protein ◽

Stop Codon ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Terminal Part ◽

Green Fluorescent ◽

Anchor Sequence

ABSTRACT Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.

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Modeling adsorption, conformation, and orientation of the Fis1 tail anchor at the mitochondrial outer membrane

10.1101/2021.06.29.450436 ◽

2021 ◽

Author(s):

Beytullah Ozgur ◽

Cory D. Dunn ◽

Mehmet Sayar

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Outer Membrane ◽

Coarse Grained ◽

Carboxyl Terminus ◽

Mitochondrial Outer Membrane ◽

Substantial Evidence ◽

Cytosolic Domain ◽

Hydrophobic Amino Acids ◽

Coarse Grained Simulations

Proteins can be targeted to organellar membranes using a tail anchor (TA), a stretch of hydrophobic amino acids found at the polypeptide carboxyl-terminus. The Fis1 protein (Fis1p), which promotes mitochondrial and peroxisomal division in the yeast Saccharomyces cerevisiae, is targeted to those organelles by its TA. Substantial evidence suggests that Fis1p insertion into the mitochondrial outer membrane can occur without the need for a translocation machinery. However, recent findings raise the possibility that Fis1p insertion into mitochondria might be promoted by a proteinaceous complex. Here, we have performed atomistic and coarse-grained simulations to analyze the adsorption, conformation and orientation of the Fis1(TA). Our results support stable insertion at the mitochondrial outer membrane in a monotopic, rather than a bitopic (transmembrane), configuration. Once inserted in the monotopic orientation, unassisted transition to the bitopic orientation is expected to be blocked by the highly charged nature of the TA carboxyl-terminus and by the Fis1p cytosolic domain. Our results are consistent with a model in which Fis1p does not require a translocation machinery for insertion at mitochondria.

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The Biogenesis Process of VDAC – From Early Cytosolic Events to Its Final Membrane Integration

Frontiers in Physiology ◽

10.3389/fphys.2021.732742 ◽

2021 ◽

Vol 12 ◽

Author(s):

Anasuya Moitra ◽

Doron Rapaport

Keyword(s):

Outer Membrane ◽

Nuclear Dna ◽

Current Knowledge ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Voltage Dependent ◽

Selective Channel ◽

Targeting Signal ◽

Cytosolic Factors

Voltage dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane. It is a membrane embedded β-barrel protein composed of 19 mostly anti-parallel β-strands that form a hydrophilic pore. Similar to the vast majority of mitochondrial proteins, VDAC is encoded by nuclear DNA, and synthesized on cytosolic ribosomes. The protein is then targeted to the mitochondria while being maintained in an import competent conformation by specific cytosolic factors. Recent studies, using yeast cells as a model system, have unearthed the long searched for mitochondrial targeting signal for VDAC and the role of cytosolic chaperones and mitochondrial import machineries in its proper biogenesis. In this review, we summarize our current knowledge regarding the early cytosolic stages of the biogenesis of VDAC molecules, the specific targeting of VDAC to the mitochondrial surface, and the subsequent integration of VDAC into the mitochondrial outer membrane by the TOM and TOB/SAM complexes.

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Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes

eLife ◽

10.7554/elife.34488 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 12

Author(s):

Daniela G Vitali ◽

Sandro Käser ◽

Antonia Kolb ◽

Kai S Dimmer ◽

Andre Schneider ◽

...

Keyword(s):

Outer Membrane ◽

Mitochondrial Biogenesis ◽

Convergent Evolution ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Topological Similarity ◽

Independent Evolution ◽

Almost All ◽

Similarity Expression

Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged.

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An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

Bioscience Reports ◽

10.1007/s10540-007-9061-0 ◽

2007 ◽

Vol 27 (6) ◽

pp. 385-401 ◽

Cited By ~ 17

Author(s):

Binks W. Wattenberg ◽

Denise Clark ◽

Stephanie Brock

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Outer Membrane ◽

Helical Structure ◽

Mitochondrial Outer Membrane ◽

Carboxy Terminus ◽

Charged Amino Acids ◽

Hydrophobic Amino Acids ◽

Signal Anchor ◽

Anchor Sequence

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.

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Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane.

The Journal of Cell Biology ◽

10.1083/jcb.126.6.1361 ◽

1994 ◽

Vol 126 (6) ◽

pp. 1361-1373 ◽

Cited By ~ 211

Author(s):

L F Sogo ◽

M P Yaffe

Keyword(s):

Outer Membrane ◽

Yeast Cells ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Morphology ◽

Wild Type ◽

Giant Mitochondria ◽

Mitochondrial Distribution ◽

Rapid Restoration ◽

Mutant Phenotypes ◽

Mutant Cells

Yeast cells with the mdm10 mutation possess giant spherical mitochondria and are defective for mitochondrial inheritance. The giant mitochondria display classical features of mitochondrial ultrastructure, yet they appear incapable of movement or division. Genetic analysis indicated that the mutant phenotypes resulted from a single nuclear mutation, and the isolated MDM10 gene restored wild-type mitochondrial distribution and morphology when introduced into mutant cells. MDM10 encodes a protein of 56.2 kD located in the mitochondrial outer membrane. Depletion of Mdm10p from cells led to a condensation of normally extended, tubular mitochondria into giant spheres, and reexpression of the protein resulted in a rapid restoration of normal mitochondrial morphology. These results demonstrate that Mdm10p can control mitochondrial morphology, and that it plays a role in the inheritance of mitochondria.

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