Mitochondrial Receptor Complex Protein

Nakai, Masaaki; Kinoshita, Kazuya; Endo, Toshiya

doi:10.1074/jbc.270.51.30571

Cited by 29 publications

(16 citation statements)

References 35 publications

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“…Although we have studied here the two distinct stages, stage A and stage B, only for the fusion protein pSu9-DHFR, the effects of deletion of the IMS domain of Tom22 were studied previously for various precursor proteins (11)(12)(13)(14). The results from the three laboratories (12)(13)(14) agree well on the observation that deletion of the IMS domain of Tom22 reduced the import of precursor proteins by 0-30%.…”

Section: Resultssupporting

confidence: 73%

“…Because transfer of the intermediate at stage B but not at stage A from the TOM complex to the TIM system is promoted by the IMS domain of Tom22, the effects of deletion of the IMS domain of Tom22 on the import of pSu9-DHFR can be used to estimate the contribution of stage B to the entire import process. In vitro import of pSu9-DHFR into mitochondria is reduced moder- (1999) ately by about Յ30% by deletion of the IMS domain of Tom22 (12)(13)(14). Because efficiency of the chase of the pSu9-DHFR intermediate from stage B was reduced to 50% by deletion of the IMS domain of Tom22, we could estimate that, at most, one-half of the pSu9-DHFR molecules are imported into the matrix via stage B (through step 4 in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The N-terminal cytosolic domain is highly acidic, and the C-terminal intermembrane space (IMS) domain contains some acidic residues, too. The roles of the cytosolic domain and the IMS domain of Tom22 in protein import have been a matter of debate (11)(12)(13)(14)(15).…”

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confidence: 99%

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Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Kanamori

Nishikawa

Nakai

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both Most mitochondrial proteins are encoded by the nuclear genome, synthesized in the cytosol as precursor proteins, and imported into mitochondria. The mitochondrial targeting signal is, in many cases, contained in the presequence, which is an N-terminal extension of the precursor protein and cleaved off by matrix-processing peptidase (MPP) after import into the matrix (1). The presequence has a potential to form a positively charged amphiphilic helix, which represents the mitochondrial targeting signal. Translocation of mitochondrial precursor proteins across the mitochondrial membranes requires coordinated actions of translocation machineries in the outer and the inner membranes, TOM (translocase of the outer membrane) and TIM (translocase of the inner membrane) complexes, respectively (2-5).In the past, many components of the TOM and TIM complexes have been identified. The TOM complex of yeast Saccharomyces cerevisiae consists of at least eight TOM proteins: Tom70, Tom37, Tom20, Tom22, Tom40, Tom5, Tom6, and Tom7. Among these TOM proteins, only Tom40 and Tom22 are essential proteins for cell growth (6-8). Tom40 is an integral membrane protein and forms a protein-conducting channel in the outer membrane (9, 10). Tom22 is anchored to the outer membrane by a central trans-membrane segment. The N-terminal cytosolic domain is highly acidic, and the C-terminal intermembrane space (IMS) domain contains some acidic residues, too. The roles of the cytosolic domain and the IMS domain of Tom22 in protein import have been a matter of debate (11-15).The question, what drives protein translocation across the outer and inner membranes in mitochondria, is one of the key issues in the protein import into mitochondria. The outer membrane lacks any transmembrane electrochemical potential or ATP-driven chaperones on the trans side of the membrane. A model was proposed in which the mitochondrial outer membrane has two binding sites for the presequence, the cis site on the cytosolic side and the trans site on the IMS side of the outer membrane (16). The transfer of the presequence from the cis site to the trans site was suggested to be coupled with unfolding of the mature domain and to drive vectorial translocation of the precursor protein across the outer membrane. Characterization has been made for the translocat...

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

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Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Kanamori

Nishikawa

Nakai

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…The positively charged character of these outer membrane targeting sequences probably allows them to utilize the same chaperones and receptors that classical mitochondrial matrix signals do, whereas the hydrophobic stretch serves as a transmembrane domain. Interestingly, the C terminus of metaxin contains six consecutive acidic residues; clusters of acidic residues in Tom22 are thought to be important in the binding of mitochondrial presequences to the trans side of the mitochondrial outer membrane during import (36), although this finding is controversial (37,38).…”

Section: Fig 4 Effect Of C-terminal Deletions On Mitochondrial Targmentioning

confidence: 99%

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Armstrong

Komiya

Bergman

et al. 1997

Journal of Biological Chemistry

123

106

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Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 3 genes in the mouse, is essential for survival of the postimplantation mouse embryo. In this study, the subcellular location, domain structure, and biochemical function of metaxin were investigated. Anti-recombinant metaxin antibodies recognized 35-and 70-kDa proteins in mitochondria from various tissues; the 35-kDa protein is consistent in size with the predicted translation product of metaxin cDNA. When metaxin cDNA was transfected into COS cells, immunofluorescence staining demonstrated that the protein is located in mitochondria. Metaxin contains a putative mitochondrial outer membrane signal anchor domain at its C terminus, and a truncated form of metaxin lacking this signal anchor domain had a reduced association with mitochondria. In addition, metaxin was highly susceptible to proteases in intact mitochondria. We therefore conclude that metaxin is a mitochondrial protein that extends into the cytosol while anchored into the outer membrane at its C terminus. In its N-terminal region, metaxin shows significant sequence identity to Tom37, a component of the outer membrane portion of the mitochondrial preprotein translocation apparatus in Saccharomyces cerevisiae, but important structural differences, including apparently different mechanisms of targeting to membranes, also exist between the two proteins. Given the similar subcellular locations of metaxin and Tom37, the possible role of metaxin in mitochondrial preprotein import was investigated. Antibodies against metaxin, when preincubated with mitochondria, partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria. Metaxin is therefore the second mammalian component of the protein translocation apparatus of the mitochondrial outer membrane to be characterized at the molecular level and the first for which an inherited mutation has been described. The early embryonic lethal phenotype of mice lacking metaxin demonstrates that efficient import of proteins into mitochondria is crucial for cellular survival. The characterization of metaxin provides an opportunity to elucidate similarities and possible differences in the mechanisms of protein import between fungi and mammals and in the phenotypes of fungi and mammals lacking mitochondrial import receptors.During studies of thrombospondin 3 (Thbs3), which encodes an extracellular matrix protein (2-4), and glucocerebrosidase (Gba), which encodes a lysosomal enzyme (5), a novel gene, termed metaxin (Mtx), was found to span the interval between Thbs3 and Gba on chromosome 3E3-F1 in the mouse (6). Mtx and Thbs3 are situated in a head-to-head orientation, with the translation start sites 1.4 kilobase pairs apart. Gba and Mtx lie in a tail-totail orientation, with the polyadenylation sites spaced 431 base pairs apart. The Mtx promoter resembles the promoters for housekeeping genes in that it is TATA-less, GC-rich, and regulated by tandem Sp1 elements within 300 base pairs of the transcription start site (7). Although ...

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“…The intermembrane spaceexposed C-terminal tail of fungal Tom22 is rich in acidic amino acid residues: the contents of acidic residues are 18.4% (net charge, Ϫ5) and 21.2% (net charge, Ϫ5) for N. crassa and S. cerevisiae, respectively. The significance of the acidic amino acid residues in this region in protein translocation, however, remains controversial (43,51,53,57). In contrast, the C-terminal tail of mammalian TOM22 does not carry net negative charges: the acidic amino acid content is 7.31% (net charge, 0).…”

Section: Figmentioning

confidence: 99%

Identification of Mammalian TOM22 as a Subunit of the Preprotein Translocase of the Mitochondrial Outer Membrane

Saeki¹,

Suzuki²,

Tsuneoka³

et al. 2000

Journal of Biological Chemistry

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A mitochondrial outer membrane protein of ϳ22 kDa (1C9-2) was purified from Vero cells assessing immunoreactivity with a monoclonal antibody, and the cDNA was cloned based on the partial amino acid sequence of the trypsin-digested fragments. 1C9-2 had 19 -20% sequence identity to fungal Tom22, a component of the preprotein translocase of the outer membrane (the TOM complex) with receptor and organizer functions. Despite such a low sequence identity, both shared a remarkable structural similarity in the hydrophobicity profile, membrane topology in the Ncyt-Cin orientation through a transmembrane domain in the middle of the molecule, and the abundant acidic amino acid residues in the N-terminal domain. The antibodies against 1C9-2 inhibited the import of a matrix-targeted preprotein into isolated mitochondria. Blue native polyacrylamide gel electrophoresis of digitonin-solubilized outer membranes revealed that 1C9-2 is firmly associated with TOM40 in the ϳ400-kDa complex, with a size and composition similar to those of the fungal TOM core complex. Furthermore, 1C9-2 complemented the defects of growth and mitochondrial protein import in ⌬tom22 yeast cells. Taken together, these results demonstrate that 1C9-2 is a functional homologue of fungal Tom22 and functions as a component of the TOM complex.Most mitochondrial proteins are encoded by the nuclear genome and are synthesized in the cytosol as preproteins. They are guided to the mitochondrial surface by cytoplasmic chaperones (1) and are then transported to the intramitochondrial compartments by the preprotein import machinery of the outer and inner membranes: the TOM 1 and TIM complexes, respectively (2-8). Extensive genetic and biochemical studies in Saccharomyces cerevisiae and Neurospora crassa have identified components of these complexes. The S. cerevisiae TOM complex is composed of at least nine proteins, Tom71, Tom70, Tom40, Tom37, Tom22, Tom20, Tom7, Tom6, and Tom5. Tom70, Tom37, Tom22, and Tom20 function as import receptors. Tom71 has strong similarity to Tom70 and is weakly associated with the TOM complex (9), although its function is unclear. Tom40 is deeply embedded in the outer membrane in a predicted ␤-barrel structure and functions as the central component of the translocation channel (10 -14). Tom6 and Tom7 modulate the dynamics of the TOM channel (15, 16). Tom5 is tightly associated with Tom40 and represents the connecting link between import receptors and the translocation channel (17). Thus, Tom40, Tom22, and three smaller Tom proteins form the general preprotein import pore (the TOM core complex) of ϳ400 kDa (14, 18). A recent study revealed that Tom22 not only functions as the import receptor, but also regulates the TOM complex organization (19). The mitochondrial inner membrane has at least two separate import machineries. The Tim23-Tim17 system mediates, in conjunction with Tim44 and mHsp70 in the matrix, mitochondrial transport of matrix-targeted preproteins (8,20). The Tim54-Tim22-Tim18 system acts with the small Tim proteins in the inter...

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Mitochondrial Receptor Complex Protein

Cited by 29 publications

References 35 publications

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Identification of Mammalian TOM22 as a Subunit of the Preprotein Translocase of the Mitochondrial Outer Membrane

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