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

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.

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

2007 ◽  
Vol 27 (6) ◽  
pp. 385-401 ◽  
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.

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 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

Comparison of large-conductance Ca(2+)-activated K+ channels in artificial bilayer and patch-clamp experiments

1994 ◽  
Vol 266 (3) ◽  
pp. C601-C610 ◽  
Author(s):  
C. L. Kapicka ◽  
A. Carl ◽  
M. L. Hall ◽  
A. L. Percival ◽  
B. W. Frey ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Apparent Distance ◽  
Bk Channels ◽  
Open Probability ◽  
K Channels ◽  
Artificial Lipid Bilayers ◽  
Excised Patches ◽  
Lipid Environment ◽  
Artificial Bilayers ◽  
Channel Properties

We compared the gating, ion conduction, and pharmacology of large-conductance Ca(2+)-activated K+ channels (BK channels) from canine colon in artificial lipid bilayers and in excised patches. Both protocols identified 270-pS K(+)-selective channels activated by depolarization and Ca2+ (approximately 130-mV shift of half-activation voltage per 10-fold change in Ca2+) that were inhibited by extracellular tetraethylammonium (TEA) and charybdotoxin. These similarities suggest that the same BK channels are studied in the two techniques. However, we found three quantitative differences between channels in artificial bilayers and patches. 1) Channels in artificial bilayers required fivefold higher free Ca2+ or 80-mV stronger depolarization for activation. 2) The voltage dependence of TEA block was smaller for channels in artificial bilayers. The apparent distance across the membrane field for the TEA binding site was 0.031 for channels in artificial bilayers and 0.23 for channels in patches. 3) ATP (2 mM) decreased open probability (Po) of channels in artificial bilayers, whereas channels in patches were unaffected. Neither GTP nor UTP reduced Po of channels in artificial bilayers. It is possible that these differences may be due to a lack of molecular identity between the channels studied in the two protocols. Alternatively, they may be attributed to alterations in channel properties during reconstitution or to influences of the artificial lipid environment.

scholarly journals Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

2005 ◽  
Vol 49 (12) ◽  
pp. 4876-4883 ◽  
Author(s):  
Axel Siroy ◽  
Virginie Molle ◽  
Christelle Lemaître-Guillier ◽  
David Vallenet ◽  
Martine Pestel-Caron ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Lipid Bilayers ◽  
Outer Membrane Protein ◽  
Carbapenem Resistance ◽  
Protein Band ◽  
Multidrug Resistant ◽  
Channel Formation ◽  
Artificial Lipid Bilayers

ABSTRACT It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M w and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.

scholarly journals Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane

2003 ◽  
Vol 160 (1) ◽  
pp. 53-64 ◽  
Author(s):  
Thomas Kaufmann ◽  
Sarah Schlipf ◽  
Javier Sanz ◽  
Karin Neubert ◽  
Reuven Stein ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Targeting ◽  
Mitochondrial Localization ◽  
Intracellular Membranes ◽  
Bona Fide ◽  
Targeting Signal ◽  
Tm Domain ◽  
Mitochondrial Targeting Sequences

It is assumed that the survival factors Bcl-2 and Bcl-xL are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-xL is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-xL requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-xL specifically functions on the MOM.

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