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  • Review Article
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The protein import motor of mitochondria

Nature Reviews Molecular Cell Biology volume 3, pages 555–565 (2002)Cite this article

Key Points

  • Transport of nuclear encoded preproteins into the mitochondria is mediated by the TOM and TIM translocases in the outer and inner mitochondrial membranes. Preproteins that are destined for the matrix carry a cleavable amino-terminal matrix targeting signal (MTS), and they are transported across TOM and TIM in a coordinated fashion and in an unfolded state. An electrical membrane potential, Δψ, is necessary for the translocation of the MTS; import of the rest of the preproteins is mediated by an ATP-driven import motor.

  • The import motor is hooked up to the outlet of the import channel of the TIM23 translocase. It consists of the peripheral inner membrane protein Tim44, the mitochondrial chaperone mtHsp70 and its co-chaperone Mge1.

  • Tim44 recruits mtHsp70s in the ATP-bound form to the import site. MtHsp70 binds a precursor that emerges from the import channel, hydrolyses ATP and is released from Tim44. The preprotein chain, in complex with mtHsp70, moves further inward. Retrograde movement is blocked by bound mtHsp70. Another mtHsp70 is binding and the precursor is fully imported by several cycles of mtHsp70 binding.

  • Two models for the mode of forward movement of preproteins have been proposed. The first — the targeted Brownian-ratchet model — involves random motion of a polypeptide chain in TOM and TIM23 channels, which is translated into vectorial movement. MtHsp70 represents an arresting component of a ratchet, which allows Brownian forward, but not backward, movement of the polypeptide chain.

  • The second model — the power-stroke model — involves a mechanical machine that pulls on the incoming polypeptide chain. A conformational change of mtHsp70 is transformed into movement that is perpendicular to the inner membrane. Tim44 serves as a fulcrum.

  • Experiments have been carried out to discriminate between the models. A preprotein with a stretch of 50 residues of glutamate residues present in a pore to which mtHsp70 cannot bind was imported into mitochondria. This indicates extensive spontaneous forward and reverse sliding in the import channels.

  • Local unfolding intermediates owing to thermal fluctuations (breathing) can be trapped by binding of denaturants, by chemical modification of amino-acid side chains or by interaction with chaperones. In this way, limited local unfolding reactions can be harvested and global unfolding can occur in the time frame of milliseconds to minutes.

  • Unfolded precursors are imported more rapidly than folded proteins. Translocation pauses when a folded domain is about to be translocated.

  • Unfolded preproteins are efficiently imported at high and low temperature. The import motor activity shows rather weak temperature dependence. By contrast, import of a preprotein that contains folded DHFR has strong temperature dependence. This indicates that the rate of spontaneous local unfolding is crucial.

  • The immunoglobulin (Ig)-like domains of muscle titin are readily imported into mitochondria although they are tightly folded. Forces of 200–300 pN are required to unfold these domains by mechanical pulling. Molecular motors like myosin or kinesin generate forces of ∼5 pN. It is unlikely that mtHsp70 could generate a force of 200 pN to unfold an Ig-like domain by a power stroke. Hydrolysis of a single ATP could generate a force of 14 pN, assuming a stroke length of 3.5 nm.

  • So, at present, experimental observations on the structure and function of the mitochondrial import motor can be explained in the framework of targeted Brownian-ratchet model. The existence of a power stroke mechanism cannot be excluded on principal grounds.

Abstract

Proteins that are destined for the matrix of mitochondria are transported into this organelle by two translocases: the TOM complex, which transports proteins across the outer mitochondrial membrane; and the TIM23 complex, which gets them through the inner mitochondrial membrane. Two models have been proposed to explain how this protein-import machinery works — a targeted Brownian ratchet, in which random motion is translated into vectorial motion, or a 'power stroke', which is exerted by a component of the import machinery. Here, we review the data for and against each model.

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Figure 1: The mitochondrial protein-import machinery.
Figure 2: Two models for unfolding and translocation of preproteins across the mitochondrial membranes.
Figure 3: Unfolding without 'pulling'.
Figure 4: Local and global unfolding of a folded protein.
Figure 5: An untargeted molecular ratchet cannot efficiently promote unfolding.

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Author information

Authors and Affiliations

  1. Institut für Physiologische Chemie, Universität München, Butenandtstraβe 5, Gebäude B, D-81377, Munich, Germany

    Walter Neupert

  2. Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, Heidelberg, 69120, Germany

    Michael Brunner

Authors
  1. Walter Neupert
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Correspondence to Walter Neupert.

Related links

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DATABASES

Interpro

DHFR domain

Ig-like domains

LocusLink

BiP

<i>Saccharomyces</i> Genome Database

D-lactate dehydrogenase

Mge1

TIM8

TIM9

TIM10

TIM12

TIM13

Tim18

TIM22

TIM23

Tim44

Tim54

Tom5

Tom6

Tom7

Tom20

Tom22

Tom40

Tom70

Swiss-Prot

DHFR

DnaJ

DnaK

Mdj1

SecA

Sec63

Sec Y translocase

titin

Glossary

TAT TRANSLOCASE

The twin-arginine translocation (TAT) system in the plasma membranes of bacteria and thylakoid membranes of chloroplasts transports folded proteins across membranes. Their sorting signals typically contain the RR motif and a further hydrophobic residue two or three residues after this motif.

SOLUTE CARRIER FAMILY

A family of structurally related inner-membrane proteins of mitochondria with six transmembrane helices that facilitate transport of cofactors and metabolites such as ATP, ADP, phosphate, citrate, malate and aspartate.

CHAPERONE

A protein that interacts transiently with unfolded segments of polypeptide chains. Chaperones help to avoid misfolding or aggregation, and support folding and/or assembly of other proteins.

BROWNIAN RATCHET

A device that can bias the Brownian motion of a particle or macromolecule in an anisotropic medium, and thereby generate a force. In the case of a Brownian protein-translocation motor, the random motion of a polypeptide chain through a membrane is biased by a non-equilibrium chemical reaction, in which a component interacts with (traps) the polypeptide for a limited time period.

STOP-TRANSFER PATHWAY

Insertion of the precursor of a membrane protein by arrest of forward movement when a transmembrane segment reaches the translocase, followed by lateral release into the lipid bilayer.

DIHYDROFOLATE REDUCTASE

(DHFR). An enzyme that catalyses the reduction of different forms of hydrofolate. These reduction reactions are linked to the reduction/oxidation of NAD+/NADH.

PASSENGER PROTEIN

A protein which, when linked to a targeting or sorting signal, is taken to the respective intracellular location, as determined by the signal. Passenger proteins help to trace the movement of proteins into organelles. In addition, specific properties of such proteins, such as stability, cofactor binding, recognition by antibodies and intrinsic fluorescence can be exploited.

J-DOMAIN

A conserved domain in DnaJ and DnaJ-like proteins, which interacts with DnaK/Hsp70 chaperones.

UREA

A denaturant that is used to promote the unfolding of proteins. Urea serves as a means to trap or 'harvest' steps of spontaneous limited unfolding or thermal fluctuations.

N-ETHYL MALEIMIDE

A sulphydryl reagent that is widely used in biochemical studies to covalently modify cysteine residues in proteins.

CO-CHAPERONE

A protein that interacts and/or cooperates with a molecular chaperone and assists in its function.

METHOTREXATE

A folate antagonist that binds to the substrate-binding site of dihydrofolate reductase and stabilizes the folded domain.

SecYEG TRANSLOCASE

A protein complex in the plasma membrane of bacteria that mediates the secretion of proteins that carry signal sequences into the periplasmic space. This translocase consists of at least eight different proteins. Translocation is driven by conformational changes of the component SecA that lead to insertion of segments of the transported protein into the translocase and require ATP hydrolysis in the bacterial cytoplasm.

Sec61 TRANSLOCASE

A protein super-complex in the membrane of the endoplasmic reticulum (ER) that mediates the translocation of other proteins into the lumen of the ER and insertion into the ER membrane. It consists of more than ten different proteins, which recognize the signal sequence and the ribosome through the signal-recognition particle, form a pore in the membrane and drive translocation that is powered by ATP hydrolysis in the lumen.

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Neupert, W., Brunner, M. The protein import motor of mitochondria. Nat Rev Mol Cell Biol 3, 555–565 (2002). https://doi.org/10.1038/nrm878

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