An Amphiphilic Lipid-Binding Domain Influences the Topology of a Signal-Anchor Sequence in the Mitochondrial Outer Membrane†

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

The Journal of Cell Biology ◽

10.1083/jcb.119.6.1451 ◽

1992 ◽

Vol 119 (6) ◽

pp. 1451-1457 ◽

Cited By ~ 83

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.

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Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619806114 ◽

2017 ◽

Vol 114 (18) ◽

pp. E3622-E3631 ◽

Cited By ~ 18

Author(s):

David P. Hoogerheide ◽

Sergei Y. Noskov ◽

Daniel Jacobs ◽

Lucie Bergdoll ◽

Vitalii Silin ◽

...

Keyword(s):

Electrochemical Impedance ◽

Physiological Role ◽

Structural Features ◽

Lipid Binding ◽

Binding Domain ◽

Water Soluble ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Membranes ◽

Dynamics Simulations ◽

Integral Proteins

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.

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

Biochemical Journal ◽

10.1042/bj20111363 ◽

2012 ◽

Vol 442 (2) ◽

pp. 381-389 ◽

Cited By ~ 14

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.

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Characterization of the N-Terminal Targeting Signal Binding Domain of the Mitochondrial Outer Membrane Receptor, Tom20†

Biochemistry ◽

10.1021/bi980746y ◽

1998 ◽

Vol 37 (38) ◽

pp. 13052-13058 ◽

Cited By ~ 18

Author(s):

Enrico Schleiff ◽

Joanne L. Turnbull

Keyword(s):

Outer Membrane ◽

Binding Domain ◽

Membrane Receptor ◽

Mitochondrial Outer Membrane ◽

Outer Membrane Receptor ◽

Targeting Signal

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Neuronal mitochondria transport Pink1 mRNA via Synaptojanin 2 to support local mitophagy

10.1101/2021.05.19.444778 ◽

2021 ◽

Author(s):

Angelika B Harbauer ◽

Simone Wanderoy ◽

J. Tabitha Hees ◽

Whitney Gibbs ◽

Martha Ordonez ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Half Life ◽

Rna Binding ◽

Binding Protein ◽

Binding Domain ◽

Mitochondrial Outer Membrane ◽

Local Translation ◽

Specific Adaptation

PTEN-induced kinase 1 (PINK1) is a very short-lived protein that is required for the removal of damaged mitochondria through Parkin translocation and mitophagy. Because the short half-life of PINK1 limits its ability to be trafficked into neurites, local translation is required for this mitophagy pathway to be active far from the soma. The Pink1 transcript is associated with and cotransported with neuronal mitochondria. In concert with translation, the mitochondrial outer membrane protein Synaptojanin 2 binding protein (SYNJ2BP) and Synaptojanin 2 (SYNJ2) are required for tethering Pink1 mRNA to mitochondria via an RNA-binding domain in SYNJ2. This neuron-specific adaptation for local translation of PINK1 provides distal mitochondria with a continuous supply of PINK1 for activation of mitophagy.

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

The Journal of Cell Biology ◽

10.1083/jcb.134.2.307 ◽

1996 ◽

Vol 134 (2) ◽

pp. 307-313 ◽

Cited By ~ 15

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.

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