Protein translocation across mitochondrial membranes

Wienhues, Ulla; Neupert, Walter

doi:10.1002/bies.950140105

Cited by 36 publications

(20 citation statements)

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“…Import of in vitro-synthesized precursor protein into isolated mitochondria. The import mixture (50 l) containing 3.0 l of the lysate and 35 S-labelled pOTC (0.8 ϫ 10 4 to 3.0 ϫ 10 4 dpm) was incubated with isolated rat liver mitochondria (100 g of protein) at 25ЊC, as described previously (23). The reaction was stopped by diluting the import mixture into 1.0 ml of the ice-cold mitochondrion isolation buffer [210 mM mannitol, 70 mM sucrose, 3 mM HEPES-KOH (pH 7.4), 0.2 mM ethylene glycol-bis(␤-aminoethyl ether)-N,N,NЈ,NЈ-tetraacetic acid (EGTA), 0.5 mg of bovine serum albumin per ml] containing 0.1 mM dinitrophenol.…”

Section: Methodsmentioning

confidence: 99%

“…Precursor proteins are loosely folded in the cytosol, bind to a mitochondrial surface receptor(s), and are then transported across the outer and inner membranes at the contact site. The presequence portions of the transported precursor proteins are proteolytically cleaved in the mitochondrial matrix, and the mature portions are folded into their native conformations (reviewed in reference 35).…”

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confidence: 99%

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Role of Heat Shock Cognate 70 Protein in Import of Ornithine Transcarbamylase Precursor into Mammalian Mitochondria

Terada

Ohtsuka

Imamoto

et al. 1995

Molecular and Cellular Biology

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The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.Most mitochondrial proteins are encoded in the nuclear genome and are synthesized on cytosolic free ribosomes as larger precursors with presequences on their amino termini. Newly synthesized precursor proteins are released into the cytosolic pool, and the half-life in the cytosol is less than a few minutes in vivo (19). Precursor proteins are loosely folded in the cytosol, bind to a mitochondrial surface receptor(s), and are then transported across the outer and inner membranes at the contact site. The presequence portions of the transported precursor proteins are proteolytically cleaved in the mitochondrial matrix, and the mature portions are folded into their native conformations (reviewed in reference 35).Studies in our laboratory (18,20) and elsewhere (22,26,27) have shown that a cytosolic factor(s) present in rabbit reticulocyte lysate or yeast cytosol is important for the import of precursor proteins into the mitochondria. Recently, mitochondrial presequence-specific cytosolic factors have been identified in rabbit reticulocyte lysate or rat liver cytosol (8,21,23,28). Other studies showed that the 70-kDa heat shock protein (hsp70) is involved in the transport of mitochondrial precursor proteins in yeasts (5, 22) and in mammals (31).h...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Role of Heat Shock Cognate 70 Protein in Import of Ornithine Transcarbamylase Precursor into Mammalian Mitochondria

Terada

Ohtsuka

Imamoto

et al. 1995

Molecular and Cellular Biology

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“…This import process has been analyzed extensively during the past decade (Glick & Schatz, 1991;Glover & Lindsay, 1992;Wienhues & Neupert, 1992). Mitochondrial targeting signals are now well characterized, especially those signals that are contained within transient N-terminal presequences (Schatz, 1987;Glick et al, 1992a).…”

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Import of cytochrome b₂ to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

et al. 1993

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Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.

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“…The Monte Carlo results are shown in Figure 2. In order to support the reliability of the simulation results, it is useful to compare them to standard Brownian theory, which requires A2 = Dozo + E&: (4) For E < E* x 1.5, the permeability rapidly decreases to 0. From our data we cannot decide whether the membrane becomes strictly impenetrable (i.e.…”

Section: Resultsmentioning

confidence: 99%

Polymer Electrophoresis across a Model Membrane

Baumgärtner¹,

Skolnick²

1994

J. Phys. Chem.

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The translocation of a polymer driven by an external bias field of strength E across a model membrane has been investigated using Monte Carlo methods. It is found that below a characteristic strength of the field E*, the membrane is practically impenetrable. In the high-field limit, E > E*, the permeability p = zdt, where zo and z are the translocation times of the polymer without and in the presence of the membrane, respectively, is independent of E. At low fields the permeability decreases according to p -exp(-E*/E). The translocation mechanism can be understood as a Kramers process describing the escape of a Brownian particle over potential barriers.

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Protein translocation across mitochondrial membranes

Cited by 36 publications

References 58 publications

Role of Heat Shock Cognate 70 Protein in Import of Ornithine Transcarbamylase Precursor into Mammalian Mitochondria

Role of Heat Shock Cognate 70 Protein in Import of Ornithine Transcarbamylase Precursor into Mammalian Mitochondria

Import of cytochrome b₂ to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

Polymer Electrophoresis across a Model Membrane

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Protein translocation across mitochondrial membranes

Cited by 36 publications

References 58 publications

Role of Heat Shock Cognate 70 Protein in Import of Ornithine Transcarbamylase Precursor into Mammalian Mitochondria

Role of Heat Shock Cognate 70 Protein in Import of Ornithine Transcarbamylase Precursor into Mammalian Mitochondria

Import of cytochrome b2 to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP

Polymer Electrophoresis across a Model Membrane

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Import of cytochrome b₂ to the mitochondrial intermembrane space: The tightly folded heme‐binding domain makes import dependent upon matrix ATP