Protein Translocation into the Intermembrane Space and Matrix of Mitochondria: Mechanisms and Driving Forces

Abstract

Mitochondria contain two aqueous subcompartments, the matrix and the intermembrane space (IMS). The matrix is enclosed by both the inner and outer mitochondrial membranes, whilst the IMS is sandwiched between the two. Proteins of the matrix are synthesized in the cytosol as preproteins, which contain amino-terminal matrix targeting sequences that mediate their translocation through translocases embedded in the outer and inner membrane. For these proteins, the translocation reaction is driven by the import motor which is part of the inner membrane translocase. The import motor employs matrix Hsp70 molecules and ATP hydrolysis to ratchet proteins into the mitochondrial matrix. Most IMS proteins lack presequences and instead utilize the IMS receptor Mia40, which facilitates their translocation across the outer membrane in a reaction that is coupled to the formation of disulfide bonds within the protein. This process requires neither ATP nor the mitochondrial membrane potential. Mia40 fulfills two roles: First, it acts as a holdase, which is crucial in the import of IMS proteins and second, it functions as a foldase, introducing disulfide bonds into newly imported proteins, which induces and stabilizes their natively folded state. For several Mia40 substrates, oxidative folding is an essential prerequisite for their assembly into oligomeric complexes. Interestingly, recent studies have shown that the two functions of Mia40 can be experimentally separated from each other by the use of specific mutants, hence providing a powerful new way to dissect the different physiological roles of Mia40. In this review we summarize the current knowledge relating to the mitochondrial matrix-targeting and the IMS-targeting/Mia40 pathway. Moreover, we discuss the mechanistic properties by which the mitochondrial import motor on the one hand and Mia40 on the other, drive the translocation of their substrates into the organelle. We propose that the lateral diffusion of Mia40 in the inner membrane and the oxidation-mediated folding of incoming polypeptides supports IMS import.

Keywords: Mia40; brownian ratchet; disulfide bond; foldase; holdase; mitochondria; protein import.

Figure 1

Mitochondria carry out a large variety of different biological activities. This figure shows some of these functions which are relevant in the context of this review: The vast amount of mitochondrial proteins need to be imported from the cytosol. (A) Proteins targeted to the IMS enter through the TOM complex and bind to the IMS receptor Mia40, which is responsible for the introduction of disulfide bonds and concomitant protein folding. (B) Proteins with an N-terminal MTS are guided to the matrix through the TOM and TIM complex. The mitochondrial processing peptidase (MPP) removes the MTSs from preproteins before they can fold into their native structures. (C) Mitochondria contain a complete genetic system for replication, transcription and translation that is entirely distinct from that in the nucleus/cytosol. The mitochondrial translation system is membrane-bound and specialized on the synthesis of a small number of very hydrophobic polypeptides. (D) Mitochondria contain a large number of metabolic enzymes that carry out a variety of catabolic and anabolic pathways. Of particular abundance and relevance are the complexes of the respiratory chain, which use the transfer of electrons to generate an electrochemical gradient which drives the synthesis of the vast majority of the cellular ATP. (E) Mitochondria interact with many other cellular compartments. The ER-mitochondria encounter structure (ERMES) tethers mitochondria to the ER, presumably to facilitate the exchange of calcium and phospholipids between their membranes. (F) The ultrastructure of mitochondria depends on the function of a number of protein complexes. Of particular importance is the “cristae organizing system” (MICOS) which participates in the formation of cristae junctions and contact sites of the inner and outer membrane. (G) Mitochondrial peptidases can regulate different mitochondrial functions through proteolytic processing and protein degradation. In addition to a number of soluble proteases, the inner membrane contains two very important ATP-hydrolysing protease complexes that belong to the AAA (ATPases associated with different cellular activities) protein family: these i-AAA and m-AAA proteases expose their proteolytic domains to the IMS and membrane sides of the inner membrane, respectively.

Figure 2

Driving forces of mitochondrial protein import. (A) Brownian ratchet: According to this model, the ATP-hydrolysis does not promote a mechanical pulling of the incoming polypeptide. Rather, Hsp70 molecules associated to Tim44 bind to unfolded segments of the translocating protein, preventing their backsliding and thus rectifying their Brownian spontaneous motion into a vectorial movement into the matrix. (B) Power stroke: It was proposed that after binding to Tim44 and the presequence of an incoming preprotein, ATP-hydrolysis in Hsp70 triggers a large conformational change within the chaperone that leads to a mechanical pulling of the preprotein into the matrix. Repeated cycles would over time drive protein-translocation in a step-wise fashion. A pulling of Hsp70 was suggested to be particularly important if a folded domain on the mitochondrial surface needs/ed to be unfolded. It should be noted that both models are not mutually exclusive. (C) Lateral Diffusion: A number of inner membrane proteins contain stop-anchor sequences just C-terminal to their presequences. How their C-terminal domains are transported across the outer membrane is not known. It was suggested that lateral diffusion thus the separation of TOM and TIM23 complexes might be critical here.

Figure 3

The disulfide relay system of the IMS. Proteins of the IMS enter the compartment through the TOM complex. They are typically of small size and contain several reduced cysteine residues. The IMS receptor/oxidoreductase Mia40 is able to form mixed disulfides with these proteins and promotes their oxidation. The FAD-containing sulfhydryl oxidase Erv1 maintains Mia40 in its oxidized form and can either transfer electrons directly to oxygen or use cytochrome c as an electron acceptor.

Figure 4

Different models of oxidative folding by Mia40. (A) Folding Trap Model: This model was inspired by the observation that reduced IMS proteins can back-translocate from the IMS to the cytosol. Since Mia40-mediated folding prevents this back-translocation, it was proposed that Mia40 does not directly promote translocation across the membrane but rather traps IMS proteins that were imported by facilitated diffusion through the TOM complex. (B) Disulfide-mediated trapping: Mia40 binds incoming proteins through mixed disulfides to prevent their backsliding into the cytosol and thus serves as a trans-site receptor that functions in a redox-mediated manner. (C) Trapping by hydrophobic binding: Mia40 is able to serve as a trans-site receptor that can mediate protein translocation in an oxidation-independent manner using hydrophobic interactions with the MIS/ITS signals in their sequence.

Figure 5

Energetics of Mia40-mediated import. (A) Lateral Diffusion: Mia40 binds to the translocating protein via hydrophobic binding which might be further stabilized by mixed disulfide bonds with the protein. Through lateral diffusion, Mia40 might drive the import reaction into the IMS. (B) Oxidation-mediated compaction: The oxidation of the incoming proteins and hence their compaction might contribute to the driving of the import process. Such a process would be limited to the import of short sequences across the TOM pore, explaining why most Mia40 substrates are of very small size.