The mitochondrial protein import motor

Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (deltapsi) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.     

Mechanisms of mitochondrial protein import.

1. Most proteins of cell organelles are synthesized as precursor proteins on cytosolic polysomes and are directed by signal sequences into the correct compartments. 2. In this review, the characteristics of mitochondrial protein uptake will be described, including the specific recognition, membrane translocation, proteolytic processing and folding of nuclear-encoded precursor proteins. 3. Recent studies indicate that a proteinaceous machinery located in the mitochondrial membranes and matrix performs these key steps of protein import.     

Versatility of the mitochondrial protein import machinery

The vast majority of mitochondrial proteins are synthesized in the cytosol and are imported into mitochondria by protein machineries located in the mitochondrial membranes. It has become clear that hydrophilic as well as hydrophobic preproteins use a common translocase in the outer mitochondrial membrane, but diverge to two distinct translocases in the inner membrane. The translocases are dynamic, high-molecular-weight complexes that have to provide specific means for the recognition of preproteins, channel formation and generation of import-driving forces.     

Control of mitochondrial protein import by pH

Protein import into mitochondria is inhibited by protons (IC(50) pH 6.5). The channels of the import machinery were examined to further investigate this pH dependence. TOM and TIM23 are the protein translocation channels of the mitochondrial outer and inner membranes, respectively, and their single channel behaviors at various pHs were determined using patch-clamp techniques. While not identical, increasing H(+) concentration decreases the open probability of both TIM23 and TOM channels. The pattern of the pH dependences of protein import and channel properties suggests TIM23 open probability can limit import of nuclear-encoded proteins into the matrix of yeast mitochondria.     

A railroad switch in mitochondrial protein import

Chacinska et al., (2005) recently clarified how translocation machineries of the mitochondrial outer and inner membranes cooperate to correctly sort preproteins destined for the mitochondrial matrix and inner membrane.     

Putting energy into mitochondrial protein import.

The import of proteins into mitochondria occurs in several steps. At least three of these steps require ATP and involve molecular chaperones. This energy requirement has served as a useful tool for elucidating the import pathways into the four mitochondrial compartments.     

In vivo study in Trypanosoma brucei links mitochondrial transfer RNA import to mitochondrial protein import

Trypanosoma brucei imports all mitochondrial transfer RNAs (tRNAs) from the cytosol. By using cell lines that allow independent tetracycline-inducible RNA interference and isopropyl-β-D-thiogalactopyranoside-inducible expression of a tagged tRNA, we show that ablation of Tim17 and mitochondrial heat-shock protein 70, components of the inner-membrane protein translocation machinery, strongly inhibits import of newly synthesized tRNAs. These findings, together with previous results in yeast and plants, suggest that the requirement for mitochondrial protein-import factors might be a conserved feature of mitochondrial tRNA import in all systems.     

Cooperation of translocase complexes in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments.     

Regulation of mitochondrial protein import by cytosolic kinases

Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria.     

The protein import machinery of the mitochondrial membranes

Mitochondria are surrounded by two membranes that contain independent and non-related protein transport machineries. Remarkable progress was recently achieved in elucidating the structure of the outer membrane import channel and in the identification of new components involved in protein traffic across the intermembrane space and the inner membrane. Traditional concepts of protein targeting and sorting had to be revised. Here we briefly summarize the data on the mitochondrial protein import system with particular emphasis on new developments and perspectives.     

Functional analysis of mitochondrial protein import in yeast

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.     

Evolution of mitochondrial protein biogenesis

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive α-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.     

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F₁ subunit of the ATP synthase (pF₁) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu²⁺ were inhibitory. Inhibition by Cu²⁺ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondria than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu²⁺ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF₁ with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu²⁺ were inhibitory. NEM, MPB, DTNB and Cu²⁺ inhibited import of the NEM-modified pF₁ into intact mitochondria. Import of pF₁ through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu²⁺ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.     

Effect of denervation-induced muscle disuse on mitochondrial protein import

This study determined whether muscle disuse affects mitochondrial protein import and whether changes in protein import are related to mitochondrial content and function. Protein import was measured using a model of unilateral peroneal nerve denervation in rats for 3 (n = 10), 7 (n = 12), or 14 (n = 14) days. We compared the import of preproteins into the matrix of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria isolated from the denervated and the contralateral control tibialis anterior muscles. Denervation led to 50% and 29% reductions in protein import after 14 days of disuse in SS and IMF mitochondria, respectively. This was accompanied by significant decreases in mitochondrial state 3 respiration, muscle mass, and whole muscle cytochrome c oxidase activity. To investigate the mechanisms involved, we assessed disuse-related changes in 1) protein import machinery components and 2) mitochondrial function, reflected by respiration and reactive oxygen species (ROS) production. Denervation significantly reduced the expression of translocases localized in the inner membrane (Tim23), outer membrane (Tom20), and mitochondrial heat shock protein 70 (mtHsp70), especially in the SS subfraction. Denervation also resulted in elevated ROS generation, and exogenous ROS was found to markedly reduce protein import. Thus our data indicate that protein import kinetics are closely related to alterations in mitochondrial respiratory capacity (r = 0.95) and are negatively impacted by ROS. Deleterious changes in the protein import system likely facilitate the reduction in mitochondrial content and the increase in organelle dysfunction (i.e., increased ROS production and decreased respiration) during chronic disuse, which likely contribute to the activation of degradative pathways leading to muscle atrophy.