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PubMed2003

Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex.

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.

Carrier Proteins↗
PubMed2000

Characterization of rat TOM40, a central component of the preprotein translocase of the mitochondrial outer membrane.

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable alpha-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a beta-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitonin-solubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a approximately 400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.

Adenosine Triphosphatases↗
PubMed2005

Taz1, an outer mitochondrial membrane protein, affects stability and assembly of inner membrane protein complexes: implications for Barth Syndrome.

The Saccharomyces cerevisiae Taz1 protein is the orthologue of human Tafazzin, a protein that when inactive causes Barth Syndrome (BTHS), a severe inherited X-linked disease. Taz1 is a mitochondrial acyltransferase involved in the remodeling of cardiolipin. We show that Taz1 is an outer mitochondrial membrane protein exposed to the intermembrane space (IMS). Transport of Taz1 into mitochondria depends on the receptor Tom5 of the translocase of the outer membrane (TOM complex) and the small Tim proteins of the IMS, but is independent of the sorting and assembly complex (SAM). TAZ1 deletion in yeast leads to growth defects on nonfermentable carbon sources, indicative of a defect in respiration. Because cardiolipin has been proposed to stabilize supercomplexes of the respiratory chain complexes III and IV, we assess supercomplexes in taz1delta mitochondria and show that these are destabilized in taz1Delta mitochondria. This leads to a selective release of a complex IV monomer from the III2IV2 supercomplex. In addition, assembly analyses of newly imported subunits into complex IV show that incorporation of the complex IV monomer into supercomplexes is affected in taz1Delta mitochondria. We conclude that inactivation of Taz1 affects both assembly and stability of respiratory chain complexes in the inner membrane of mitochondria.

Acyltransferases↗
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↗
PubMed1999

The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore.

Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex). This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane. This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.

Adenosine Triphosphatases↗
PubMed1999

Inactivation of the Neurospora crassa mitochondrial outer membrane protein TOM70 by repeat-induced point mutation (RIP) causes defects in mitochondrial protein import and morphology.

Mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles. Recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the TOM complex (translocase of the outer mitochondrial membrane). The largest component of this complex is TOM70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. To investigate the functional role of TOM70 in Neurospora crassa the tom70 gene was inactivated using the natural phenomenon of repeat-induced point mutation (RIP). Mutant strains were identified that harbored RIPed tom70 alleles and contained no immunologically detectable TOM70. Strains that lack TOM70 grow more slowly than wild-type strains, conidiate poorly, and contain enlarged mitochondria. In vitro preprotein import studies using TOM70-deficient mitochondria revealed a defect in the uptake of the ADP/ATP carrier. Other preproteins tested were imported at wild-type rates with the exception of the precursor of the mitochondrial-processing peptidase (MPP) which was imported more efficiently by TOM70-deficient mitochondria. These data support the view that TOM70 plays a role as a specific receptor for carrier proteins in mitochondrial-preprotein import. The presence of tetratricopeptide repeats (TPRs) in the TOM70 sequence and the enlarged shape of mitochondria lacking TOM70 raise the possibility that the protein also plays a role in the maintenance of mitochondrial morphology.

Cell Division↗
PubMed2004

Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila.

A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.

Amino Acid Sequence↗
PubMed2006

The C-terminal TPR domain of Tom70 defines a family of mitochondrial protein import receptors found only in animals and fungi.

In fungi and animals the translocase in the outer mitochondrial membrane (TOM complex) consists of multiple components including the receptor subunit Tom70. Genome sequence analyses suggest no Tom70 receptor subunit exists in plants or protozoans, raising questions about its ancestry, function and the importance of its activity. Here we characterise the relationships within the Tom70 family of proteins. We find that in both fungi and animals, a conserved domain structure exists within the Tom70 family, with a transmembrane segment followed by 11 tetratricopeptide repeat motifs organised in three distinct domains. The C-terminal domain of Tom70 is highly conserved, and crucial for the import of hydrophobic substrate proteins, including those with and those without N-terminal presequences. Tom70 likely arose after fungi and animals diverged from other eukaryote lineages including plants, and subsequent gene duplication gave rise to a paralogue specific to the Saccharomyces group of yeasts. In animals and in fungi, Tom70 plays a fundamental role in the import of precursor proteins, by assisting relatively hydrophobic regions of substrate proteins into the translocation channel in the outer mitochondrial membrane. Proteins that function equivalently to Tom70 may have arisen independently in plants and protists.

Animals↗
PubMed2006

Crystal structure of yeast mitochondrial peripheral membrane protein Tim44p C-terminal domain.

The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of TIM23 translocon. Tim44p can tightly associate with the inner mitochondrial membrane. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, we have determined the crystal structure of the yeast Tim44p C-terminal domain to 3.2A resolution using the MAD method. The Tim44p C-terminal domain forms a monomer in the crystal structure and contains six alpha-helices and four antiparallel beta-strands. A large hydrophobic pocket was identified on the Tim44p structure surface. The N-terminal helix A1 is positively charged and the helix A1 protrudes out from the Tim44p main body.

Amino Acid Sequence↗
PubMed2000

Protein translocation pathways of the mitochondrion.

The biogenesis of mitochondria depends on the coordinated import of precursor proteins from the cytosol coupled with the export of mitochondrially coded proteins from the matrix to the inner membrane. The mitochondria contain an elaborate network of protein translocases in the outer and inner membrane along with a battery of chaperones and processing enzymes in the matrix and intermembrane space to mediate protein translocation. A mitochondrial protein, often with an amino-terminal targeting sequence, is escorted through the cytosol by chaperones to the TOM complex (translocase of the outer membrane). After crossing the outer membrane, the import pathway diverges; however, one of two TIM complexes (translocase of inner membrane) is generally utilized. This review is focused on the later stages of protein import after the outer membrane has been crossed. An accompanying paper by Lithgow reviews the early stages of protein translocation.

Animals↗
PubMed2002

Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
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↗
PubMed2002

The protein import motor of mitochondria.

Proteins that are destined for the matrix of mitochondria are transported into this organelle by two translocases: the TOM complex, which transports proteins across the outer mitochondrial membrane; and the TIM23 complex, which gets them through the inner mitochondrial membrane. Two models have been proposed to explain how this protein-import machinery works -- a targeted Brownian ratchet, in which random motion is translated into vectorial motion, or a 'power stroke', which is exerted by a component of the import machinery. Here, we review the data for and against each model.

Membrane Proteins↗
PubMed2004

Assembling the mitochondrial outer membrane.

The general preprotein translocase of the outer mitochondrial membrane (TOM complex) transports virtually all mitochondrial precursor proteins, but cannot assemble outer-membrane precursors into functional complexes. A recently discovered sorting and assembly machinery (SAM complex) is essential for integration and assembly of outer-membrane proteins, revealing unexpected connections to mitochondrial evolution and morphology.

Adenosine Triphosphate↗
PubMed1998

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22.

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.

Biological Transport↗
PubMed2004

Sam35 of the mitochondrial protein sorting and assembly machinery is a peripheral outer membrane protein essential for cell viability.

The mitochondrial outer membrane contains two integral proteins essential for cell viability, Tom40 of the translocase of the outer membrane (TOM complex) and Sam50 of the sorting and assembly machinery (SAM complex). Here we report the identification of Sam35, the first peripheral mitochondrial outer membrane protein that is essential for cell viability. Sam35 (encoded by the Saccharomyces cerevisiae ORF YHR083w) is a novel subunit of the SAM complex and is crucial for the assembly pathway of outer membrane beta-barrel proteins, such as the precursors of Tom40 and porin. Sam35 is not required for the import of inner membrane or matrix targeted proteins. The presence of two essential proteins in the SAM complex, Sam35 and Sam50, indicates that it plays a central role in mitochondrial biogenesis.

Amino Acid Sequence↗
PubMed2004

Membrane-embedded C-terminal segment of rat mitochondrial TOM40 constitutes protein-conducting pore with enriched beta-structure.

TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% beta-sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(DeltaN165)) as a recombinant protein with 62% beta-structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched beta-structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.

Animals↗