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At least 19 recordsLinked to original sources
PubMed2004

Mitochondrial protein import. Requirement of presequence elements and tom components for precursor binding to the TOM complex.

Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (the cis site) and on the intermembrane space side (the trans site). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to the cis and trans sites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F(0)-ATPase are required for binding to the trans site. The interaction between the presequence and the cis site is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to the trans site and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.

Membrane Proteins↗
PubMed2005

Dissection of the mitochondrial import and assembly pathway for human Tom40.

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.

Adenosine Triphosphate↗
PubMed2002

Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons.

Import of nuclear-encoded mitochondrial proteins requires the action of at least two different import machines, called translocons, in the mitochondrial inner membrane (IM). The TIM23 complex mediates the translocation of proteins into the mitochondria matrix, whereas the TIM22 complex is required for the insertion of polytopic proteins into the IM. While the two translocons are distinct and composed of separate subunits, the essential reactions in each complex are carried out by homologous proteins. In addition, the core components of both the TIM23 and TIM22 translocons have been shown to form aqueous pores in the mitochondrial IM. In this review, we summarize what is known about import of proteins across the mitochondrial IM.

Intracellular Membranes↗
PubMed2002

Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex.

The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures.

Fungal Proteins↗
PubMed2003

Mitochondrial biogenesis and the role of the protein import pathway.

PURPOSE: The importance of the mitochondrial protein import pathway, discussed relative to other steps involved in the overall biogenesis of the organelle, are reviewed. RESULTS: Mitochondrial biogenesis is a product of complex interactions between the nuclear and mitochondrial genomes. Signaling pathways, such as those activated by exercise, initiate the activation of transcription factors that increase the production of mRNA from nuclear and mitochondrial DNA. Nuclear gene products are translated in the cytosol as precursor proteins with inherent targeting signals. These precursor proteins interact with molecular chaperones that direct them to the import machinery of the outer membrane (Tom complex). The precursor is unfolded and transferred through the outer membrane, across the intermembrane space to the mitochondrial inner membrane translocases (Tim complex). Intramitochondrial components (mtHSP70) pull the precursor into the matrix, cleave off the targeting sequence (mitochondrial processing peptidase), and refold the protein (HSP60, cpn10) into its mature conformation. Physiological stressors such as contractile activity and thyroid hormone accelerate protein import into the mitochondria, coincident with an increase in the expression of some components of the import machinery. This is important for the overall expansion of the mitochondrial reticulum. Conversely, impairments in the import process can be a cause of mitochondrial dysfunction and disease. CONCLUSIONS: Efforts to further characterize the components of the import machinery, to define the role of specific machinery components on the import rate, and to examine protein import function in a variety of mitochondrial diseases are warranted.

Carrier Proteins↗
PubMed1997

Interactions of the human mitochondrial protein import receptor, hTom20, with precursor proteins in vitro reveal pleiotropic specificities and different receptor domain requirements.

Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membrane of the mitochondrion. To analyze the contribution of Tom20 to precursor protein recognition, the cytosolic domain of the human mitochondrial import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase and conditions established to measure specific interactions of the receptor component with precursor proteins in vitro. Reconstitution of receptor binding from purified components revealed that a prototypic matrix-destined precursor protein, pODHFR, interacts with Tom20 by a mechanism that is dependent on an active matrix targeting signal but does not require cytosolic components or ATP. Binding was influenced by both salt concentration and detergent. The effect of salt or detergent, however, varied for different precursor proteins. In particular, detergent selectively enhanced binding of pODHFR to receptor, possibly because of induced changes in the structure of the signal sequence. Finally, mutations were introduced into hTom20 which had a dramatic effect on binding of some precursor proteins but not on others. Taken together, the results suggest that hTom20 recognizes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity for these precursor proteins may involve different domains within the receptor molecule.

Adenosine Triphosphate↗
PubMed2002

Functions of outer membrane receptors in mitochondrial protein import.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.

Amino Acid Sequence↗
PubMed2003

Import of nuclear-encoded proteins into mitochondria.

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria through protein translocation machineries of the outer and inner membranes. In this review, we discuss the arrangement of the various translocation complexes and the function of individual import components. We also outline the various targeting pathways that preproteins can take in order to reach their appropriate sub-mitochondrial compartment.

Animals↗
PubMed2002

Mitochondrial import of the ADP/ATP carrier: the essential TIM complex of the intermembrane space is required for precursor release from the TOM complex.

The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.

Intracellular Membranes↗
PubMed2004

The J domain-related cochaperone Tim16 is a constituent of the mitochondrial TIM23 preprotein translocase.

Mitochondria import the vast majority of their proteins from the cytosol. The mitochondrial import motor of the TIM23 translocase drives the translocation of precursor proteins across the outer and inner membrane in an ATP-dependent reaction. Tim44 at the inner face of the translocation pore recruits the chaperone mtHsp70, which binds the incoming precursor protein. This reaction is assisted by the cochaperones Tim14 and Mge1. We have identified a novel essential cochaperone, Tim16. It is related to J-domain proteins and forms a stable subcomplex with the J protein Tim14. Depletion of Tim16 has a marked effect on protein import into the mitochondrial matrix, impairs the interaction of Tim14 with the TIM23 complex and leads to severe structural changes of the import motor. In conclusion, Tim16 is a constituent of the TIM23 preprotein translocase, where it exerts crucial functions in the import motor.

Fungal Proteins↗
PubMed2002

Delivery of nascent polypeptides to the mitochondrial surface.

Thousands of polypeptides with diverse biochemical properties, some of which are extremely hydrophobic, are targeted from cytoplasmic ribosomes to the surface of mitochondria. Localised synthesis, as well as transient interactions with a wide array of molecular chaperones and other cytoplasmic factors, can promote productive interaction of mitochondrial proteins with the TOM complex to initiate protein import into mitochondria.

Carrier Proteins↗
PubMed1998

Identification of the protein import components of the rat mitochondrial inner membrane, rTIM17, rTIM23, and rTIM44.

We cloned rat liver mitochondrial 18.1, 21.9, and 51.0 kDa proteins with a significant structural homology to the components of the translocase of the yeast mitochondrial inner membrane, Tim17, Tim23, and Tim44. The 18.1 and 21.9 kDa proteins were synthesized as mature forms having four potential transmembrane segments and localized to the mitochondrial inner membrane. The 51.0 kDa protein is a precursor having a presequence of approximately 6 kDa which is cleaved during import into the mitochondria. The mature 45 kDa protein is located in the matrix, both in a soluble form and in a membrane-bound, alkali-extractable form. Immunofluorescence microscopy confirmed the location of all three proteins in the mitochondria. Antibodies against the 21.9 kDa protein, but not those against the 18.1 and 51.0 kDa proteins, inhibited the precursor import into the mitoplasts in vitro. Immunoprecipitation indicated that all three proteins interacted with the protein in transit to the matrix. Immunoprecipitation also revealed that the 18.1 kDa protein formed a complex with the 21.9 kDa protein and the 45 kDa protein with mHsp70; the latter complex was dissociated in an ATP- or ADP-dependent manner and the reaction was impeded by AMP-PNP or inorganic phosphate. These assays thus demonstrated the 18.1, 21.9, and 45 kDa proteins to be the translocator components of the rat mitochondrial inner membrane and, therefore, the functional homologues of Tim17, Tim23, and Tim44, respectively.

Amino Acid Sequence↗
PubMed2004

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.

Carrier Proteins↗
PubMed2005

Import of rat mitochondrial citrate carrier (CIC) at increasing salt concentrations promotes presequence binding to import receptor Tom20 and inhibits membrane translocation.

Mitochondria contain a family of related carrier proteins that mediate transport of metabolites across the mitochondrial inner membrane. All members of this family are synthesized in the cytosol. We characterized the interactions of newly synthesized rat citrate carrier (CIC) precursor protein (pCIC) with the components of the mitochondrial protein import machinery. pCIC contains both a positively charged presequence of 13 amino acids and internal targeting sequences. We found that the pCIC presequence does not interfere with the import pathway and merely acts as an internal chaperone in the cytosol. Under conditions of increased ionic strength, the pCIC presequence binds to the import receptor Tom20 and accumulates at the mitochondrial surface, thereby delaying pCIC translocation across the mitochondrial outer membrane. Similarly, the presequence of the bovine phosphate carrier (PiC) precursor protein (pPiC) is arrested at the mitochondrial surface when salt concentrations are elevated. We conclude that presequences can only act as mediators of mitochondrial protein import if they allow rapid release from import receptor sites. Release from receptors sites may be rate-limiting in translocation.

Animals↗
PubMed2006

Characterization of Mmp37p, a Saccharomyces cerevisiae mitochondrial matrix protein with a role in mitochondrial protein import.

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

Cell Survival↗