scholarly journals The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery.

1996 ◽  
Vol 134 (2) ◽  
pp. 307-313 ◽  
Author(s):  
H M McBride ◽  
I S Goping ◽  
G C Shore
Keyword(s):  
Outer Membrane ◽  
Protein Translocation ◽  
Mitochondrial Outer Membrane ◽  
Mammalian Mitochondria ◽  
Precursor Proteins ◽  
Targeting Signal ◽  
Signal Anchor ◽  
Positively Charged ◽  
Import Receptor

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal-anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein-translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.

scholarly journals Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei

2017 ◽  
Vol 292 (8) ◽  
pp. 3400-3410 ◽  
Author(s):  
Julia Bruggisser ◽  
Sandro Käser ◽  
Jan Mani ◽  
André Schneider
Keyword(s):  
Outer Membrane ◽  
Trypanosoma Brucei ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Transgenic Cells ◽  
Membrane Spanning ◽  
Necessary And Sufficient ◽  
Inducible Rnai ◽  
Positively Charged

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei, is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not.

scholarly journals Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

1998 ◽  
Vol 18 (6) ◽  
pp. 3173-3181 ◽  
Author(s):  
Frank E. Nargang ◽  
Doron Rapaport ◽  
R. Gary Ritzel ◽  
Walter Neupert ◽  
Roland Lill
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Wild Type ◽  
Binding Studies ◽  
Amino Terminal ◽  
Precursor Proteins ◽  
Mutant Strains ◽  
Cytosolic Domain ◽  
Charged Residues ◽  
Positively Charged

ABSTRACT TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing atom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutanttom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors

2012 ◽  
Vol 442 (2) ◽  
pp. 381-389 ◽  
Author(s):  
Elisa Merklinger ◽  
Yana Gofman ◽  
Alexej Kedrov ◽  
Arnold J. M. Driessen ◽  
Nir Ben-Tal ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid Bilayers ◽  
Lipid Composition ◽  
Helical Structure ◽  
Mitochondrial Outer Membrane ◽  
Artificial Lipid Bilayers ◽  
Targeting Signal ◽  
Signal Anchor ◽  
Lipid Environment ◽  
Cytosolic Factors

The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an α-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide's affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.

scholarly journals Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

2001 ◽  
Vol 154 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Andreas Hiltbrunner ◽  
Jörg Bauer ◽  
Pierre-Alexandre Vidi ◽  
Sibylle Infanger ◽  
Petra Weibel ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Large Scale ◽  
Protein Import ◽  
Protein Translocation ◽  
Soluble Form ◽  
Chloroplast Biogenesis ◽  
Outer Envelope ◽  
Precursor Proteins ◽  
Outer Envelope Membrane ◽  
Import Receptor

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

scholarly journals Genome-wide screens in yeast highlight a role for cardiolipin in biogenesis of mitochondrial outer membrane multispan proteins

2015 ◽  
pp. MCB.00107-15 ◽  
Author(s):  
Julia Sauerwald ◽  
Tobias Jores ◽  
Michal Eisenberg-Bord ◽  
Silvia Gabriela Chuartzman ◽  
Maya Schuldiner ◽  
...  
Keyword(s):  
Outer Membrane ◽  
De Novo ◽  
Mitochondrial Outer Membrane ◽  
Special Group ◽  
Mitochondrial Inner Membrane ◽  
Genome Wide ◽  
Steady State Levels ◽  
Lower Capacity ◽  
Import Receptor

A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the Mitochondrial Import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in yeast. Cardiolipin synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4 and Om14 in mitochondria fromcrd1Δcells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower capacity to importin vitronewly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtainde novosynthesized cardiolipin (CL) molecules. Hence, our findings suggest CL as an important component in the biogenesis of MOM multispan proteins.

scholarly journals A signal-anchor sequence selective for the mitochondrial outer membrane.

1992 ◽  
Vol 119 (6) ◽  
pp. 1451-1457 ◽  
Author(s):  
H M McBride ◽  
D G Millar ◽  
J M Li ◽  
G C Shore
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Hybrid Protein ◽  
Transmembrane Segment ◽  
Structural Domains ◽  
Signal Anchor ◽  
Anchor Sequence ◽  
Rate Limiting

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

scholarly journals Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence.

1993 ◽  
Vol 268 (34) ◽  
pp. 25265-25268 ◽  
Author(s):  
M Nguyen ◽  
D G Millar ◽  
V W Yong ◽  
S J Korsmeyer ◽  
G C Shore
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Signal Anchor ◽  
Anchor Sequence

scholarly journals Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins

2013 ◽  
Vol 288 (23) ◽  
pp. 16451-16459 ◽  
Author(s):  
Thomas Becker ◽  
Susanne E. Horvath ◽  
Lena Böttinger ◽  
Natalia Gebert ◽  
Günther Daum ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Proper Function ◽  
Mitochondrial Outer Membrane ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Helical Proteins ◽  
The Stability

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.

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 Bacterial tail anchors can target to the mitochondrial outer membrane

2017 ◽  
Author(s):  
Güleycan Lutfullahoğlu Bal ◽  
Abdurrahman Keskin ◽  
Ayşe Bengisu Seferoğlu ◽  
Cory D. Dunn
Keyword(s):  
Outer Membrane ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Mitochondrial Outer Membrane ◽  
Bacterial Proteins ◽  
Rate Limiting Step ◽  
Protein Entry ◽  
Rate Limiting

ABSTRACTDuring the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was refashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol. Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM. Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

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