Signal recognition initiates reorganization of the presequence translocase during protein import.

The mitochondrial presequence translocase interacts with presequence-containing precursors at the intermembrane space (IMS) side of the inner membrane to mediate their translocation into the matrix. Little is known as too how these matrix-targeting signals activate the translocase in order to initiate precursor transport. Therefore, we analysed how signal recognition by the presequence translocase initiates reorganization among Tim-proteins during import. Our analyses revealed that the presequence receptor Tim50 interacts with Tim21 in a signal-sensitive manner in a process that involves the IMS-domain of the Tim23 channel. The signal-driven release of Tim21 from Tim50 promotes recruitment of Pam17 and thus triggers formation of the motor-associated form of the TIM23 complex required for matrix transport.

Motor-free mitochondrial presequence translocase drives membrane integration of preproteins.

The mitochondrial inner membrane is the central energy-converting membrane of eukaryotic cells. The electrochemical proton gradient generated by the respiratory chain drives the ATP synthase. To maintain this proton-motive force, the inner membrane forms a tight barrier and strictly controls the translocation of ions. However, the major preprotein transport machinery of the inner membrane, termed the presequence translocase, translocates polypeptide chains into or across the membrane. Different views exist of the molecular mechanism of the translocase, in particular of the coupling with the import motor of the matrix. We have reconstituted preprotein transport into the mitochondrial inner membrane by incorporating the purified presequence translocase into cardiolipin-containing liposomes. We show that the motor-free form of the presequence translocase integrates preproteins into the membrane. The reconstituted presequence translocase responds to targeting peptides and mediates voltage-driven preprotein translocation, lateral release and insertion into the lipid phase. Thus, the minimal system for preprotein integration into the mitochondrial inner membrane is the presequence translocase, a cardiolipin-rich membrane and a membrane potential.

On the mechanism of preprotein import by the mitochondrial presequence translocase.

Mitochondria are organelles of endosymbiontic origin that contain more than one thousand different proteins. The vast majority of these proteins is synthesized in the cytosol and imported into one of four mitochondrial subcompartments: outer membrane, intermembrane space, inner membrane and matrix. Several import pathways exist and are committed to different classes of precursor proteins. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) mediates import of precursor proteins with cleavable amino-terminal presequences. Presequences direct precursors across the inner membrane. The combination of this presequence with adjacent regions determines if a precursor is fully translocated into the matrix or laterally sorted into the inner mitochondrial membrane. The membrane-embedded TIM23(SORT) complex mediates the membrane potential-dependent membrane insertion of precursor proteins with a stop-transfer sequence downstream of the mitochondrial targeting signal. In contrast, translocation of precursor proteins into the matrix requires the recruitment of the presequence translocase-associated motor (PAM) to the TIM23 complex. This ATP-driven import motor consists of mitochondrial Hsp70 and several membrane-associated co-chaperones. These two structurally and functionally distinct forms of the TIM23 complex (TIM23(SORT) and TIM23(MOTOR)) are in a dynamic equilibrium with each other. In this review, we discuss recent advances in our understanding of the mechanisms of matrix translocation and membrane insertion by the TIM23 machinery.

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis.

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.

Distinct forms of mitochondrial TOM-TIM supercomplexes define signal-dependent states of preprotein sorting.

Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23(SORT)). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23(SORT) and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import.

Mitochondrial protein import motor: differential role of Tim44 in the recruitment of Pam17 and J-complex to the presequence translocase.

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane translocase.

Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain.

The mitochondrial presequence translocase transports preproteins to either matrix or inner membrane. Two different translocase forms have been identified: the matrix transport form, which binds the heat-shock protein 70 (Hsp70) motor, and the inner membrane-sorting form, which lacks the motor but contains translocase of inner mitochondrial membrane 21 (Tim21). The sorting form interacts with the respiratory chain in a Tim21-dependent manner. It is unknown whether the respiratory chain-bound translocase transports preproteins and how the switch between sorting form and motor form occurs. We report that the respiratory chain-bound translocase contains preproteins in transit and, surprisingly, not only sorted but also matrix-targeted preproteins. Presequence translocase-associated motor (Pam) 16 and 18, two regulatory components of the six-subunit motor, interact with the respiratory chain independently of Tim21. Thus, the respiratory chain-bound presequence translocase is not only active in preprotein sorting to the inner membrane but also in an early stage of matrix translocation. The motor does not assemble en bloc with the translocase but apparently in a step-wise manner with the Pam16/18 module before the Hsp70 core.

Tim50 maintains the permeability barrier of the mitochondrial inner membrane.

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.