scholarly journals Identification of the Signal Directing Tim9 and Tim10 into the Intermembrane Space of Mitochondria

2009 ◽  
Vol 20 (10) ◽  
pp. 2530-2539 ◽  
Author(s):  
Dusanka Milenkovic ◽  
Thomas Ramming ◽  
Judith M. Müller ◽  
Lena-Sophie Wenz ◽  
Natalia Gebert ◽  
...  
Keyword(s):  
Amino Acid ◽  
Outer Membrane ◽  
Sufficient Information ◽  
Intermembrane Space ◽  
Amino Acid Residues ◽  
Sorting Signal ◽  
Precursor Proteins ◽  
Mitochondrial Intermembrane Space ◽  
Substrate Proteins

The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space.

scholarly journals Reconstituted TOM Core Complex and Tim9/Tim10 Complex of Mitochondria Are Sufficient for Translocation of the ADP/ATP Carrier across Membranes

2004 ◽  
Vol 15 (3) ◽  
pp. 1445-1458 ◽  
Author(s):  
Andreja Vasiljev ◽  
Uwe Ahting ◽  
Frank E. Nargang ◽  
Nancy E. Go ◽  
Shukry J. Habib ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid Vesicles ◽  
Intermembrane Space ◽  
Core Complex ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Solute Carrier ◽  
Membrane Peptide ◽  
Membrane Spanning ◽  
Substrate Proteins

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

scholarly journals A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria

2014 ◽  
Vol 25 (25) ◽  
pp. 3999-4009 ◽  
Author(s):  
Agnieszka Gornicka ◽  
Piotr Bragoszewski ◽  
Piotr Chroscicki ◽  
Lena-Sophie Wenz ◽  
Christian Schulz ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Membrane ◽  
Alternative Pathway ◽  
Intermembrane Space ◽  
Outer Mitochondrial Membrane ◽  
Membrane Translocation ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Core Components

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

scholarly journals Precursor Oxidation by Mia40 and Erv1 Promotes Vectorial Transport of Proteins into the Mitochondrial Intermembrane Space

2008 ◽  
Vol 19 (1) ◽  
pp. 226-236 ◽  
Author(s):  
Judith M. Müller ◽  
Dusanka Milenkovic ◽  
Bernard Guiard ◽  
Nikolaus Pfanner ◽  
Agnieszka Chacinska
Keyword(s):  
Experimental Evidence ◽  
Cysteine Residues ◽  
Intermembrane Space ◽  
Disulfide Formation ◽  
Precursor Proteins ◽  
Mitochondrial Intermembrane Space ◽  
Vectorial Transport ◽  
Tim Proteins

The mitochondrial intermembrane space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential intermembrane space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of intermembrane space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. Whereas the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the intermembrane space. Thus, oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial intermembrane space.

scholarly journals Mitochondrial Import of the ADP/ATP Carrier: the Essential TIM Complex of the Intermembrane Space Is Required for Precursor Release from the TOM Complex

2002 ◽  
Vol 22 (22) ◽  
pp. 7780-7789 ◽  
Author(s):  
Kaye N. Truscott ◽  
Nils Wiedemann ◽  
Peter Rehling ◽  
Hanne Müller ◽  
Chris Meisinger ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Intermembrane Space ◽  
Hydrophobic Membrane ◽  
Tom Complex ◽  
Native Electrophoresis ◽  
Mitochondrial Intermembrane Space ◽  
First Time ◽  
Blue Native Electrophoresis ◽  
Step Mechanism ◽  
Blue Native

ABSTRACT The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.

scholarly journals Structural and Functional Roles of the Surface-Exposed Loops of the β-Barrel Membrane Protein OmpA fromEscherichia coli

1999 ◽  
Vol 181 (12) ◽  
pp. 3688-3694 ◽  
Author(s):  
Ralf Koebnik
Keyword(s):  
Amino Acid ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Outer Membrane ◽  
High Efficiency ◽  
Outer Membrane Proteins ◽  
Protein Domain ◽  
Amino Acid Residues ◽  
Functional Roles ◽  
Ompa Protein

ABSTRACT The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel β-barrel whose eight transmembrane β-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral β-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest β-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.

scholarly journals Identification of amino acid residues required for ferric-anguibactin transport in the outer-membrane receptor FatA of Vibrio anguillarum

Microbiology ◽  
2007 ◽  
Vol 153 (2) ◽  
pp. 570-584 ◽  
Author(s):  
Claudia S López ◽  
Alejandro F Alice ◽  
Ranjan Chakraborty ◽  
Jorge H Crosa
Keyword(s):  
Amino Acid ◽  
Outer Membrane ◽  
Vibrio Anguillarum ◽  
Membrane Receptor ◽  
Amino Acid Residues ◽  
Outer Membrane Receptor

Mutations in a 19-amino-acid hydrophobic region of the yeast cytochrome c1 presequence prevent sorting to the mitochondrial intermembrane space

1992 ◽  
Vol 12 (10) ◽  
pp. 4677-4686
Author(s):  
R E Jensen ◽  
S Schmidt ◽  
R J Mark
Keyword(s):  
Amino Acid ◽  
Intermediate Form ◽  
Intermembrane Space ◽  
Protein Amino Acid ◽  
Hydrophobic Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytochrome C1 ◽  
The Matrix ◽  
Hydrophobic Amino Acids ◽  
Mitochondrial Intermembrane Space

Most mitochondrial proteins destined for the intermembrane space (IMS) carry in their presequence information for localization to the IMS in addition to information for their import. By selecting for mutants in the yeast Saccharomyces cerevisiae that mislocalize an IMS-targeted fusion protein, we identified mutations in the IMS sorting signal of the cytochrome c1 protein. Amino acid substitutions or deletions in a stretch of 19 hydrophobic amino acids of the cytochrome c1 presequence resulted in accumulation of the intermediate form of the cytochrome c1 protein in the matrix. In some cases, the accumulated intermediate appeared to be slowly exported from the matrix, across the inner membrane to the IMS. Our results support the hypothesis that the cytochrome c1 precursor is normally imported completely into the matrix and then exported to the IMS.

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.

scholarly journals Retro-translocation of mitochondrial intermembrane space proteins

2015 ◽  
Vol 112 (25) ◽  
pp. 7713-7718 ◽  
Author(s):  
Piotr Bragoszewski ◽  
Michal Wasilewski ◽  
Paulina Sakowska ◽  
Agnieszka Gornicka ◽  
Lena Böttinger ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Proteins ◽  
Dynamic Processes ◽  
Driving Mechanism ◽  
Intermembrane Space ◽  
Unfolded Proteins ◽  
Mitochondrial Proteome ◽  
Assembly Pathway ◽  
Mitochondrial Intermembrane Space ◽  
Pore Protein

The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.

Sign in / Sign up

Export Citation Format

Share Document