A role for Tim21 in membrane-potential-dependent preprotein sorting in mitochondria

The mitochondrial inner membrane harbors complexes of the respiratory chain and translocase complexes for preproteins. The membrane potential generated by the respiratory chain is essential for ATP production by the mitochondrial ATP synthase and as a driving force for protein import. It is generally believed that the preprotein translocases just use the membrane potential without getting into physical contact with respiratory-chain complexes. Here, we show that the presequence translocase interacts with the respiratory chain. Tim21, a specific subunit of the sorting-active presequence translocase , recruits proton-pumping respiratory-chain complexes and stimulates preprotein insertion. Thus, the presequence translocase cooperates with the respiratory chain and promotes membrane-potential-dependent protein sorting into the inner mitochondrial membrane. These findings suggest a new coupling mechanism in an energy-transducing membrane.     

Pam16 has an essential role in the mitochondrial protein import motor

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.     

The Mitochondrial ADP/ATP Carrier Associates with the Inner Membrane Presequence Translocase in a Stoichiometric Manner

The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low.     

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.     

Tim23 links the inner and outer mitochondrial membranes

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.     

Structural basis for the function of Tim50 in the mitochondrial presequence translocase

Many mitochondrial proteins are synthesized as preproteins carrying amino-terminal presequences in the cytosol. The preproteins are imported by the translocase of the outer mitochondrial membrane and the presequence translocase of the inner membrane. Tim50 and Tim23 transfer preproteins through the intermembrane space to the inner membrane. We report the crystal structure of the intermembrane space domain of yeast Tim50 to 1.83 Å resolution. A protruding β-hairpin of Tim50 is crucial for interaction with Tim23, providing a molecular basis for the cooperation of Tim50 and Tim23 in preprotein translocation to the protein-conducting channel of the mitochondrial inner membrane.     

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space

The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.     

Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preprotein-binding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.     

The Channel-Forming Sym1 Protein Is Transported by the TIM23 Complex in a Presequence-Independent Manner

The majority of multispanning inner mitochondrial membrane proteins utilize internal targeting signals, which direct them to the carrier translocase (TIM22 complex), for their import. MPV17 and its Saccharomyces cerevisiae orthologue Sym1 are multispanning inner membrane proteins of unknown function with an amino-terminal presequence that suggests they may be targeted to the mitochondria. Mutations affecting MPV17 are associated with mitochondrial DNA depletion syndrome (MDDS). Reconstitution of purified Sym1 into planar lipid bilayers and electrophysiological measurements have demonstrated that Sym1 forms a membrane pore. To address the biogenesis of Sym1, which oligomerizes in the inner mitochondrial membrane, we studied its import and assembly pathway. Sym1 forms a transport intermediate at the translocase of the outer membrane (TOM) complex. Surprisingly, Sym1 was not transported into mitochondria by an amino-terminal signal, and in contrast to what has been observed in carrier proteins, Sym1 transport and assembly into the inner membrane were independent of small translocase of mitochondrial inner membrane (TIM) and TIM22 complexes. Instead, Sym1 required the presequence of translocase for its biogenesis. Our analyses have revealed a novel transport mechanism for a polytopic membrane protein in which internal signals direct the precursor into the inner membrane via the TIM23 complex, indicating a presequence-independent function of this translocase.     

A MICOS–TIM22 Association Promotes Carrier Import into Human Mitochondria

Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.     

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.     

The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.     

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.     

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23.

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.     

Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes

Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23^IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import.     

Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.     

Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex

N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.     

The assembly pathway of the mitochondrial carrier translocase involves four preprotein translocases

The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.     

A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix- targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt- hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt- hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.