An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.     

Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c-myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug-treated c-myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine-specific modifying agent revealed that Bax inserted both the 'pore domain' (helices alpha5-alpha6), and the tail-anchor (helix alpha9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc-expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore-forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.     

Role of MINOS in Mitochondrial Membrane Architecture: Cristae Morphology and Outer Membrane Interactions Differentially Depend on Mitofilin Domains

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.     

The intermembrane space domain of Tim23 is intrinsically disordered with a distinct binding region for presequences

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N‐terminal, soluble domain in the intermembrane space (IMS) and a C‐terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N‐terminal domain of Tim23, Tim23N (residues 1–96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71–84. In a charge‐hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network.     

Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. Although these morphologic changes are considered crucial for cellular functions, the underlying mechanisms remain elusive, especially in mammalian cells. We characterized two rat mitochondrial outer membrane proteins, Mfn1 and Mfn2, with distinct tissue expressions, that are homologous to Drosophila Fzo, a GTPase involved in mitochondrial fusion. Expression of the GTPase-domain mutant of Mfn2 (Mfn2(K109T)) in HeLa cells induced mitochondrial fragmentation in which Mfn2(K109T) localized at the restricted domains. Immuno-electronmicroscopy revealed that Mfn2(K109T) was concentrated at the contact domains between adjacent mitochondria, suggesting that fusion of the outer membrane was arrested at some intermediate step. Mfn1 expression induced highly connected tubular network structures depending on the functional GTPase domain. The Mfn1-induced tubular networks were suppressed by co-expression with Mfn2. In vivo depletion of either isoform by RNA interference revealed that both are required to maintain normal mitochondrial morphology. The fusion of differentially-labeled mitochondria in HeLa cells subjected to depletion of either Mfn isoform and subsequent cell fusion by hemagglutinating virus of Japan revealed that both proteins have distinct functions in mitochondrial fusion. We conclude that the two Mfn isoforms cooperate in mitochondrial fusion in mammalian cells.     

Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex

The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of β-barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of β-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of β-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds β-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of β-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.     

Functional definition of outer membrane proteins involved in preprotein import into mitochondria

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.     

Targeting and Import Mechanism of Coiled-coil Helix Coiled-coil Helix Domain-containing Protein 3 (ChChd3) into the Mitochondrial Intermembrane Space

Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX₉C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys¹⁹³, and the active site cysteine in Mia40, Cys⁵⁵. Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.     

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.     

Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.     

Protein secretion through autotransporter and two-partner pathways

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.     

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.     

Versatility of the mitochondrial protein import machinery

The vast majority of mitochondrial proteins are synthesized in the cytosol and are imported into mitochondria by protein machineries located in the mitochondrial membranes. It has become clear that hydrophilic as well as hydrophobic preproteins use a common translocase in the outer mitochondrial membrane, but diverge to two distinct translocases in the inner membrane. The translocases are dynamic, high-molecular-weight complexes that have to provide specific means for the recognition of preproteins, channel formation and generation of import-driving forces.     

The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion

In yeast, mitochondrial fusion requires Ugo1p and two GTPases, Fzo1p and Mgm1p. Ugo1p is anchored in the mitochondrial outer membrane with its N terminus facing the cytosol and C terminus in the intermembrane space. Fzo1p is also an outer membrane protein, whereas Mgm1p is located in the intermembrane space. Recent studies suggest that these three proteins form protein complexes that mediate mitochondrial fusion. Here, we show that the cytoplasmic domain of Ugo1p directly interacts with Fzo1p, whereas its intermembrane space domain binds to Mgm1p. We identified the Ugo1p-binding site in Fzo1p and demonstrated that Ugo1p-Fzo1p interaction is essential for the formation of mitochondrial shape, maintenance of mitochondrial DNA, and fusion of mitochondria. Although the GTPase domains of Fzo1p and Mgm1p regulate mitochondrial fusion, they were not required for association with Ugo1p. Furthermore, we found that Ugo1p bridges the interaction between Fzo1p and Mgm1p in mitochondria. Our data indicate that distinct regions of Ugo1p bind directly to Fzo1p and Mgm1p and thereby link these two GTPases during mitochondrial fusion.     

Biogenesis of mitochondrial outer membrane proteins

Mitochondria are surrounded by two distinct membranes: the outer and the inner membrane. The mitochondrial outer membrane mediates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Proteins of this membrane are nuclear-encoded and synthesized as precursor proteins in the cytosol. They are targeted to the mitochondria and inserted into their target membrane via various pathways. This review summarizes our current knowledge of the sorting signals for this specific targeting and describes the mechanisms by which the mitochondrial import machineries recognize precursor proteins, mediate their membrane integration and facilitate assembly into functional complexes.     

A dynamic machinery for import of mitochondrial precursor proteins

Mitochondria contain approximately 1000 different proteins, which are located in four different compartments, outer membrane, inner membrane, intermembrane space and matrix. The vast majority of these proteins has to be imported from the cytosol. Therefore, sophisticated molecular machineries have evolved that mediate protein translocation across or insertion into mitochondrial membranes and subsequent assembly into multi-subunit complexes. While the initial entry of virtually all mitochondrial proteins is mediated by the general import pore of the outer membrane, at least four different downstream pathways are dedicated to import and assembly of proteins into a specific compartment.     

The N-terminal domain of Tob55 has a receptor-like function in the biogenesis of mitochondrial beta-barrel proteins

beta-Barrel proteins constitute a distinct class of mitochondrial outer membrane proteins. For import into mitochondria, their precursor forms engage the TOM complex. They are then relayed to the TOB complex, which mediates their insertion into the outer membrane. We studied the structure-function relationships of the core component of the TOB complex, Tob55. Tob55 precursors with deletions in the N-terminal domain were not affected in their targeting to and insertion into the mitochondrial outer membrane. Replacement of wild-type Tob55 by these deletion variants resulted in reduced growth of cells, and mitochondria isolated from such cells were impaired in their capacity to import beta-barrel precursors. The purified N-terminal domain was able to bind beta-barrel precursors in a specific manner. Collectively, these results demonstrate that the N-terminal domain of Tob55 recognizes precursors of beta-barrel proteins. This recognition may contribute to the coupling of the translocation of beta-barrel precursors across the TOM complex to their interaction with the TOB complex.     

Signal-anchored proteins follow a unique insertion pathway into the outer membrane of mitochondria

Signal-anchored proteins are a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. Like the vast majority of mitochondrial proteins, signal-anchored proteins are synthesized on cytosolic ribosomes and are subsequently imported into the organelle. We have studied the mechanisms by which precursors of these proteins are recognized by the mitochondria and are inserted into the outer membrane. The import of signal-anchored proteins was found to be independent of the known import receptors, Tom20 and Tom70, but to require the major Tom component, Tom40. In contrast to precursors destined to internal compartments of mitochondria and those of outer membrane beta-barrel proteins, precursors of signal-anchored proteins appear not to be inserted via the general import pore. Taken together, we propose a novel pathway for insertion of these proteins into the outer membrane of mitochondria.     

Complex formation and turnover of mitochondrial transporters and ion channels

Mitochondria are responsible for many vital cellular functions in eukaryotic cells, such as ATP production, steroid synthesis and prosthetic group biogenesis. The vital functions of mitochondria are possible due to the compartmental nature of this organelle. Mitochondria form a dynamic network that can exist as a network throughout a cell or as distinct individual structures. Mitochondria are also composed of two membranes, an inner and outer membrane. The inner mitochondrial membrane (IMM) is significantly larger than the outer membrane and must fold upon itself to be contained within the outer mitochondrial membrane (OMM). These folds are known as cristae. Altogether these different membrane compartments specialize in different functions of the mitochondria. The OMM is responsible for passage of small metabolites into and out of the mitochondria while excluding macromolecules. The IMM is a highly selective barrier between the solutes of the cytosol and those within the mitochondrial matrix. Cristae specialize in oxidative phosphorylation. The functions of these membranes are afforded by membrane proteins that are able to transport specific solutes. The appropriate localization, assembly into multi-subunit protein complexes, and wild-type function of these membrane proteins therefore is vital for mitochondria to maintain appropriate function and support cellular survival. This review will address the composition and functions of mitochondrial membrane localized multi-subunit protein complexes along with how these proteins undergo degradation to maintain homeostatic functions of mitochondria in the context of mitochondria specific transporters and ion channels. Due to the large number of known mitochondrial membrane transporters and ion channels this review will focus on the topics presented at the Mitochondrial Ion Channels and Transporters Symposium hosted by the New York University College of Dentistry in September 2015 in honor of Casey Kinnally.