scholarly journals A unified model for the biogenesis of mitochondrial outer membrane multi-span proteins

2021 ◽  
Author(s):  
Jialin Zhou ◽  
Martin Jung ◽  
Kai S. Dimmer ◽  
Doron Rapaport
Keyword(s):  
Phosphatidic Acid ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Lipid Phase ◽  
Yeast Mitochondria ◽  
Mitochondrial Import ◽  
Membrane Behavior ◽  
Double Deletion ◽  
Variable Extent ◽  
Import Receptor

The mitochondrial outer membrane (MOM) harbors proteins that traverse the membrane via several helical segments, so-called multi-span proteins. Two contradicting mechanisms were suggested to describe their integration into the MOM. The first proposes that the mitochondrial import (MIM) complex facilitates this process and functions as an insertase, whereas the second suggests that such proteins can integrate into the lipid phase without the assistance of import factors in a process that is enhanced by phosphatidic acid. To resolve this discrepancy and obtain new insights on the biogenesis of these proteins, we addressed this issue using yeast mitochondria and the multi-span protein Om14. Testing different truncation variants, we show that only the full-length protein contains all the required information that assure targeting specificity. Employing a specific insertion assay and several single and double deletion strains, we show that neither the import receptor Tom70 nor any other protein with a cytosolically exposed domain have a crucial contribution to the biogenesis process. We further demonstrate that Mim1 and Porin are required for optimal membrane integration of Om14 but none of them is absolutely required. Unfolding of the newly synthesized protein, its optimal hydrophobicity, as well as higher fluidity of the membrane dramatically enhanced the import capacity of Om14. Collectively, our findings suggest that MOM multi-span proteins can follow different biogenesis pathways in which proteinaceous elements and membrane behavior contribute to a variable extent to the combined efficiency.

scholarly journals Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway

2011 ◽  
Vol 194 (3) ◽  
pp. 397-405 ◽  
Author(s):  
Dražen Papić ◽  
Katrin Krumpe ◽  
Jovana Dukanovic ◽  
Kai S. Dimmer ◽  
Doron Rapaport
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Gel Electrophoresis ◽  
Mitochondrial Outer Membrane ◽  
Yeast Mitochondria ◽  
Membrane Complex ◽  
Native Gel ◽  
Insertion Process ◽  
Import Receptor

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.

scholarly journals Human mitochondrial import receptor Tom70 functions as a monomer

2010 ◽  
Vol 429 (3) ◽  
pp. 553-563 ◽  
Author(s):  
Anna C. Y. Fan ◽  
Lisandra M. Gava ◽  
Carlos H. I. Ramos ◽  
Jason C. Young
Keyword(s):  
Heat Shock ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Size Exclusion ◽  
Mitochondrial Outer Membrane ◽  
Cross Linking ◽  
Wild Type ◽  
Oligomeric State ◽  
Mitochondrial Import ◽  
Import Receptor

The mitochondrial import receptor Tom70 (translocase of the mitochondrial outer membrane 70) interacts with chaperone–preprotein complexes through two domains: one that binds Hsp70 (heat-shock protein 70)/Hsc70 (heat-shock cognate 70) and Hsp90, and a second that binds preproteins. The oligomeric state of Tom70 has been controversial, with evidence for both monomeric and homodimeric forms. In the present paper, we report that the functional state of human Tom70 appears to be a monomer with mechanistic implications for its function in mitochondrial protein import. Based on analytical ultracentrifugation, cross-linking, size-exclusion chromatography and multi-angle light scattering, we found that the soluble cytosolic fragment of human Tom70 exists in equilibrium between monomer and dimer. A point mutation introduced at the predicted dimer interface increased the percentage of monomeric Tom70. Although chaperone docking to the mutant was the same as to the wild-type, the mutant was significantly more active in preprotein targeting. Cross-linking also demonstrated that the mutant formed stronger contacts with preprotein. However, cross-linking of full-length wild-type Tom70 on the mitochondrial membrane showed little evidence of homodimers. These results indicate that the Tom70 monomers are the functional form of the receptor, whereas the homodimers appear to be a minor population, and may represent an inactive state.

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

1995 ◽  
Vol 15 (6) ◽  
pp. 3382-3389 ◽  
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.

scholarly journals Multiple 40-kDa Heat-Shock Protein Chaperones Function in Tom70-dependent Mitochondrial Import

2007 ◽  
Vol 18 (9) ◽  
pp. 3414-3428 ◽  
Author(s):  
Melanie K. Bhangoo ◽  
Stefan Tzankov ◽  
Anna C.Y. Fan ◽  
Kurt Dejgaard ◽  
David Y. Thomas ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Outer Membrane ◽  
Adenine Nucleotide ◽  
Dominant Negative ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Import ◽  
Specific Subset ◽  
Reticulocyte Lysate ◽  
Dependent Protein

Mitochondrial preproteins that are imported via the translocase of the mitochondrial outer membrane (Tom)70 receptor are complexed with cytosolic chaperones before targeting to the mitochondrial outer membrane. The adenine nucleotide transporter (ANT) follows this pathway, and its purified mature form is identical to the preprotein. Purified ANT was reconstituted with chaperones in reticulocyte lysate, and bound proteins were identified by mass spectrometry. In addition to 70-kDa heat-shock cognate protein (Hsc70) and 90-kDa heat-shock protein (Hsp90), a specific subset of cochaperones were found, but no mitochondria-specific targeting factors were found. Interestingly, three different Hsp40-related J-domain proteins were identified: DJA1, DJA2, and DJA4. The DJAs bound preproteins to different extents through their C-terminal regions. DJA dominant-negative mutants lacking the N-terminal J-domains impaired mitochondrial import. The mutants blocked the binding of Hsc70 to preprotein, but with varying efficiency. The DJAs also showed significant differences in activation of the Hsc70 ATPase and Hsc70-dependent protein refolding. In HeLa cells, the DJAs increased new protein folding and mitochondrial import, although to different extents. No single DJA was superior to the others in all aspects, but each had a profile of partial specialization. The Hsp90 cochaperones p23 and Aha1 also regulated Hsp90–preprotein interactions. We suggest that multiple cochaperones with similar yet partially specialized properties cooperate in optimal chaperone–preprotein complexes.

scholarly journals OM14 is a mitochondrial receptor for cytosolic ribosomes that supports co-translational import into mitochondria

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Chen Lesnik ◽  
Yifat Cohen ◽  
Avigail Atir-Lande ◽  
Maya Schuldiner ◽  
Yoav Arava
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Strong Support ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Import ◽  
Translational Mode ◽  
Nascent Chain ◽  
Cytosolic Ribosomes

Abstract It is well established that import of proteins into mitochondria can occur after their complete synthesis by cytosolic ribosomes. Recently, an additional model was revived, proposing that some proteins are imported co-translationally. This model entails association of ribosomes with the mitochondrial outer membrane, shown to be mediated through the ribosome-associated chaperone nascent chain-associated complex (NAC). However, the mitochondrial receptor of this complex is unknown. Here, we identify the Saccharomyces cerevisiae outer membrane protein OM14 as a receptor for NAC. OM14Δ mitochondria have significantly lower amounts of associated NAC and ribosomes, and ribosomes from NAC[Δ] cells have reduced levels of associated OM14. Importantly, mitochondrial import assays reveal a significant decrease in import efficiency into OM14Δ mitochondria, and OM14-dependent import necessitates NAC. Our results identify OM14 as the first mitochondrial receptor for ribosome-associated NAC and reveal its importance for import. These results provide a strong support for an additional, co-translational mode of import into mitochondria.

scholarly journals Biogenesis of Tom40, Core Component of the Tom Complex of Mitochondria

1999 ◽  
Vol 146 (2) ◽  
pp. 321-332 ◽  
Author(s):  
Doron Rapaport ◽  
Walter Neupert
Keyword(s):  
Outer Membrane ◽  
Initial Step ◽  
Terminal Segment ◽  
Mitochondrial Outer Membrane ◽  
Core Component ◽  
Core Element ◽  
The Core ◽  
Tom Complex ◽  
Partially Folded Conformation ◽  
Import Receptor

Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH2 nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH2-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.

Characterization of Different Reactive Lysines in Bovine Heart Mitochondrial Porin

2002 ◽  
Vol 383 (12) ◽  
pp. 1967-1970 ◽  
Author(s):  
J.A. Al jamal
Keyword(s):  
Outer Membrane ◽  
Fluorescein Isothiocyanate ◽  
Mitochondrial Outer Membrane ◽  
Lipid Phase ◽  
Lipid Bilayer Membranes ◽  
Electrical Measurements ◽  
Elution Pattern ◽  
Outer Membrane Porin ◽  
Lysine Residues ◽  
Substantial Change

Abstract Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channels selectivity and its voltage dependence.

scholarly journals Dissecting the interactions of PINK1 with the TOM complex in depolarized mitochondria

2022 ◽  
Author(s):  
Klaudia Maruszczak ◽  
Martin Jung ◽  
Shafqat Rasool ◽  
Jean-Francois Trempe ◽  
Doron Rapaport
Keyword(s):  
Parkinson’S Disease ◽  
Outer Membrane ◽  
Molecular Mechanism ◽  
Mitochondrial Outer Membrane ◽  
Structural Protein ◽  
Mitochondria Dysfunction ◽  
Tom Complex ◽  
The Individual ◽  
Import Receptor

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinson's disease. PINK1, which is mutated in some cases of familiar Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.

scholarly journals The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

2015 ◽  
Vol 210 (6) ◽  
pp. 951-960 ◽  
Author(s):  
F.-Nora Vögtle ◽  
Michael Keller ◽  
Asli A. Taskin ◽  
Susanne E. Horvath ◽  
Xue Li Guan ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Phosphatidic Acid ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Lithocholic Acid ◽  
Mitochondrial Protein ◽  
Phospholipid Composition ◽  
Mitochondrial Proteins ◽  
Mitochondrial Outer Membrane

Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.

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