Tim23 Links the Inner and Outer Mitochondrial Membranes (original) (raw)
Cell, 2002
Ahting et al., 2001). The TOM Nagoya University complex also contains a presequence binding site (the Chikusa-ku, Nagoya 464-8602 trans site) for mitochondrial proteins on the IMS side of Japan the outer membrane, which consists of the IMS side 2 Department of Biochemistry and Cell Biology region of Tom40 and is close to the IMS domain of Institute of Gerontology Tom22 (Mayer et al., 1995; Rapaport et al., 1997; Kana-Nippon Medical School mori et al., 1999). Kosugi-cho, Nakahara-ku, Kawasaki The mitochondrial inner membrane contains two dis-Kanagawa 211-8533 tinct translocator complexes, the TIM22 complex and Japan the TIM23 complex, for protein insertion into and across the inner membrane. The TIM22 complex is required for insertion of presequence-less polytopic membrane Summary proteins including mitochondrial carrier proteins into the inner membrane (Koehler et al., 1999; Bauer et al., 2000). Based on the results of site-specific photocrosslinking The TIM23 complex mediates translocation across and of translocation intermediates, we have identified Tim50, insertion into the inner membrane of presequence-cona component of the yeast TIM23 import machinery, taining precursor proteins (Bauer et al., 2000; Pfanner which mediates translocation of presequence-conand Geissler, 2001). The TIM23 complex consists of taining proteins across the mitochondrial inner mempolytopic integral membrane proteins, Tim23 and Tim17, brane. Tim50 is anchored to the inner mitochondrial and a peripheral membrane protein, Tim44, associated membrane, exposing the C-terminal domain to the inwith Ssc1p, Hsp70 in the matrix (mtHsp70). Tim23 can termembrane space. Tim50 interacts with the N-terform a voltage-gated protein conducting channel (Bauer minal intermembrane space domain of Tim23. Funcet al., 1996; Truscott et al., 2001) and functions as a tional defects of Tim50 either by depletion of the receptor for presequences (Bauer et al., 1996; Komiya protein or addition of anti-Tim50 antibodies block the et al., 1998). Translocation of the presequence of mitoprotein translocation across the inner membrane. A chondrial precursor proteins requires the membrane potranslocation intermediate accumulated at the TOM tential (⌬⌿) across the inner membrane, whereas uncomplex is crosslinked to Tim50. We suggest that folding and transmembrane movement of the mature Tim50, in cooperation with Tim23, facilitates transfer part require the ATP-dependent reaction cycle of of the translocating protein from the TOM complex to mtHsp70 in cooperation with Tim44 and Yge1p (Mge1p) the TIM23 complex in the matrix (Pfanner and Geissler, 2001). The translocation of precursor proteins across the Introduction inner membrane appears to be tightly linked to their translocation across the outer membrane, because no Mitochondria consist of the outer and inner membranes soluble intermediates in the IMS can be observed and two aqueous compartments, the intermembrane (Schleyer and Neupert, 1985; Kanamori et al., 1997). space (IMS) and the matrix. Most mitochondrial proteins Nevertheless, the TOM complex in the outer membrane are encoded by the nuclear genome, synthesized in the and the TIM23 complex in the inner membrane are not cytosol, and subsequently imported into mitochondria. permanently linked, but they can interact only in the Once within the mitochondria, the imported mitochonpresence of a translocating precursor protein (Berthold drial proteins are sorted to one of the four submitochonet al., 1995; Horst et al., 1995; Dekker et al., 1997). Theredrial compartments. Targeting and sorting information fore, a precursor protein translocating through the TOM is often contained in the N-terminal cleavable presecomplex needs to engage the TIM23 complex in the quence of mitochondrial precursor proteins themselves, boundary inner membrane, which is closely apposed to while it can also be contained in the mature part of the outer membrane, to form a transient "translocation mitochondrial proteins as internal signals. The complex contact site" (Pfanner et al., 1992). Although the IMS processes of mitochondrial protein import and sorting domain of Tom22 and the N-terminal segment of Tim23 are mediated by elaborate systems in mitochondria, inappear to facilitate the transfer of the translocating precluding the translocator complexes in the outer and cursor protein from the TOM complex to the TIM23 cominner mitochondrial membranes (Neupert, 1997; Pfanner plex (Court et al., 1996; Moczko et al., 1997; Kanamori and Geissler, 2001). et al., 1999; Donzeau et al., 2000), the molecular mecha-The translocator of the outer mitochondrial membrane nism underlying the link of the translocation across the (TOM) mediates the entry of probably all nuclear enouter membrane and that across the inner membrane remains unclear. In the present study, we identified Tim50, a subunit of 3 Correspondence: endo@biochem.chem.nagoya-u.ac.jp Cell 520 Cell 528 James, P., Halladay, J., and Craig, E.A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425-1436. Kanamori, T., Nishikawa, S., Shin, I., Schultz, G., and Endo, T. (1997). Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking. Proc. Natl. Acad. Sci. USA 94, 485-490. Kanamori, T., Nishikawa, S., Nakai, M., Shin, I., Schultz, P.G., and Endo, T. (1999). Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane. Proc. Natl. Acad. Sci. USA 96, 3634-3639. Koehler, C.M., Merchant, S., and Schatz, G. (1999). How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem. Sci. 24, 428-432. Komiya, T., Rospert, S., Koehler, C., Looser, R., Schatz, G., and Mihara, K. (1998). Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis. EMBO J. 17, 3886-3898.
Journal of Biological Chemistry, 2002
Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an ϳ120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable ϳ380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis.
The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes
EMBO reports, 2006
Proteins destined for the mitochondrial matrix are imported by the translocase of the outer membrane-the TOM complex-and the presequence translocase of the inner membrane-the TIM23 complex. At present, there is no structural information on components of the presequence translocase. Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor. We crystallized the binding domain of Tim21 of Saccharomyces cerevisiae and determined its structure at 1.6 Å resolution. The Tim21 structure represents a new a/b-mixed protein fold with two a-helices flanked by an extended eight-stranded b-sheet. We also identified a core sequence of Tom22 that binds to Tim21. Furthermore, negatively charged amino-acid residues of Tom22 are important for binding to Tim21. Here we suggest a mechanism for the TOM-TIM interaction.
F1000 - Post-publication peer review of the biomedical literature, 2002
im23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH 2-terminal targeting presequence that are imported in a linear NH 2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p-Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p-Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p-Tim13p complex T is not dependent on zinc, and the purified Tim8p-Tim13p complex does not coordinate zinc in the conserved twin CX 3 C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH 2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.
On the mechanism of preprotein import by the mitochondrial presequence translocase
Biochimica Et Biophysica Acta-molecular Cell Research, 2010
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.
The Preprotein Translocation Channel of the Outer Membrane of Mitochondria
Cell, 1998
In the outer membrane, the TOM com-Goethestrasse 33 plex mediates the recognition of preproteins, their trans-80336 Mü nchen fer through the outer membrane, and the insertion of Germany resident outer membrane proteins. The inner membrane 2 Department of Biological Sciences translocation machinery (TIM) facilitates further transfer University of Alberta of preproteins across and into the inner membrane. Two Edmonton, Alberta T6G 2E9 TIM complexes exist that are specific for different sub-Canada sets of preproteins (
Journal of Biological Chemistry, 2006
The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.
The TOM Core Complex: The General Protein Import Pore of the Outer Membrane of Mitochondria
The Journal of Cell Biology, 1999
Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of ∼2.1 nm and a height of ∼7 nm. Tom40 is the key structu...