Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria.

The BCS1 protein is anchored in the mitochondrial inner membrane via a single transmembrane domain and has an N(out)-C(in) topology. Unlike the majority of nuclear encoded mitochondrial preproteins, the BCS1 protein does not contain an N-terminal targeting sequence. A positively charged segment of amino acids which is located immediately C-terminal to the transmembrane domain acts as an internal targeting signal. In order to function, we postulate that this sequence co-operates with the transmembrane domain to form a tight hairpin loop structure. This loop is translocated across the inner membrane via the MIM/mt-Hsp70 machinery in a membrane potential-dependent manner. This novel mechanism of import and sorting of the BCS1 protein is proposed to represent a more general mechanism used by a number of inner membrane proteins.     

Targeting of the 22 kDa integral peroxisomal membrane protein

Investigating targeting of the 22 kDa peroxisomal membrane protein (Pmp22p) to the peroxisomal membrane we have confined the targeting signal to amino acid residues 16-37 located in the N-terminal cytoplasmic tail. Comparison of Pmp22p orthologous sequences revealed a conserved motif Y3xL3xP3x(KQN) which might represent the core of this targeting signal not found so far in other Pmps. Fusion of the Pmp22p N-terminal tail to the C-terminal portion of Pmp22p which per se is not targeted to peroxisomes, conveys peroxisomal targeting. These data suggest that Pmp22p is targeted to peroxisomes by a new membrane targeting signal which is necessary and sufficient to target a polypeptide containing two transmembrane spans to peroxisomes.     

Signal-anchor domains of proteins of the outer membrane of mitochondria: structural and functional characteristics

We have studied the topogenesis of 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. To determine the role of these latter sequences in the targeting and insertion of such proteins we took two approaches. First, a functional complementation assay was used to define the structural elements that together with the anchor domain make up the topogenic signal. Moderate hydrophobicity of the transmembrane domain was found to be the most important requirement. Variants with a scrambled sequence of the membrane-spanning segment were only partially functional suggesting that specificity in the amino acid sequence is also of considerable importance. A net positive charge at both flanking regions of the transmembrane domain contributes to the efficiency of targeting and membrane integration but is not an essential structural feature of this signal. Second, chimeras of Tom20, Tom70, and OM45 were generated that contained the cytosolic domain of Tom20 or Tom70 and the anchor domain of one of the other members of the class. These hybrid proteins were able to rescue the growth of cells lacking Tom20 or Tom70. Thus, anchor domains of outer membrane proteins are functionally interchangeable. They play only a minor role in the specific function of these proteins, but have a decisive role in topogenic signaling.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Analysis of dinoflagellate mitochondrial protein sorting signals indicates a highly stable protein targeting system across eukaryotic diversity

Protein targeting into mitochondria from the cytoplasm is fundamental to the cell biology of all eukaryotes. Our understanding of this process is heavily biased towards “model” organisms, such as animals and fungi, and it is less clear how conserved this process is throughout diverse eukaryotes. In this study, we have surveyed mitochondrial protein sorting signals from a representative of the dinoflagellate algae. Dinoflagellates are a phylum belonging to the group Alveolata, which also includes apicomplexan parasites and ciliates. We generated 46 mitochondrial gene sequences from the dinoflagellate Karlodinium micrum and analysed these for mitochondrial sorting signals. Most of the sequences contain predicted N-terminal peptide extensions that conform to mitochondrial targeting peptides from animals and fungi in terms of length, amino acid composition, and propensity to form amphipathic α-helices. The remainder lack predicted mitochondrial targeting peptides and represent carrier proteins of the inner mitochondrial membrane that have internal targeting signals in model eukaryotes. We tested for functional conservation of the dinoflagellate mitochondrial sorting signals by expressing K. micrum mitochondrial proteins in the fungus Saccharomyces cerevisiae. Both the N-terminal and internal targeting signals were sufficiently conserved to operate in this distantly related system. This study indicates that the character of mitochondrial sorting signals was well established prior to the radiation of major eukaryotic lineages and has shown remarkable conservation during long periods of evolution.     

Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP.

In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0-ATPase (Su9) from Neurospora crassa, an integral inner membrane protein. A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1-112)-DHFR). This protein attains the correct topology across the inner membrane (Nout-Cin) following import. The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N-terminal tail. Import and export steps can be experimentally dissected into two distinct events. Translocation of the N-terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain. Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis. Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop-transfer signal. Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors.     

Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.     

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.     

Switching the sorting mode of membrane proteins from cotranslational endoplasmic reticulum targeting to posttranslational mitochondrial import

Hydrophobic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, mediated by hydrophobic signal sequence. Mitochondrial membrane proteins escape this mechanism despite their hydrophobic character. We examined sorting of membrane proteins into the mitochondria, by using mitochondrial ATP-binding cassette (ABC) transporter isoform (ABC-me). In the absence of 135-residue N-terminal hydrophilic segment (N135), the membrane domain was integrated into the ER membrane in COS7 cells. Other sequences that were sufficient to import soluble protein into mitochondria could not import the membrane domain. N135 imports other membrane proteins into mitochondria. N135 prevents cotranslational targeting of the membrane domain to ER and in turn achieves posttranslational import into mitochondria. In a cell-free system, N135 suppresses targeting to the ER membranes, although it does not affect recognition of hydrophobic segments by signal recognition particle. We conclude that the N135 segment blocks the ER targeting of membrane proteins even in the absence of mitochondria and switches the sorting mode from cotranslational ER integration to posttranslational mitochondrial import.     

Mmm1p spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation

In the yeast Saccharomyces cerevisiae, the integral membrane protein Mmm1p is required for maintenance of mitochondrial morphology and retention of mitochondrial DNA (mtDNA). Mmm1p localizes to discrete foci on mitochondria that are adjacent to mtDNA nucleoids in the matrix, raising the possibility that this protein plays a direct role in organizing, replicating, or segregating mtDNA. Although Mmm1p has been shown to cross the outer membrane with its C terminus facing the cytoplasm, the location of the N terminus has not been resolved. Here we show that Mmm1p spans both the outer and inner mitochondrial membranes, exposing its N terminus to the matrix. Surprisingly, deletion of the N-terminal extension decreased steady-state levels of the Mmm1 protein but did not affect mitochondrial morphology or mtDNA maintenance. Moreover, expression of Neurospora crassa MMM1, which naturally lacks a long N-terminal extension, substituted for loss of Mmm1p in budding yeast. These results indicate that the matrix-exposed portion of Mmm1p is not essential for mtDNA nucleoid maintenance. Additional studies revealed that the transmembrane segment and C-terminal domain of Mmm1p are required for foci formation and mitochondrial targeting, respectively. Our data suggest that the double membrane-spanning topology of Mmm1p at the membrane contact site is critical for formation of tubular mitochondria.     

The essential function of Tim12 in vivo is ensured by the assembly interactions of its C-terminal domain

The small Tims chaperone hydrophobic precursors across the mitochondrial intermembrane space. Tim9 and Tim10 form the soluble TIM10 complex that binds precursors exiting from the outer membrane. Tim12 functions downstream, as the only small Tim peripherally attached on the inner membrane. We show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, in contrast to the other small Tims. We find that the C-terminal end of Tim12 is essential in vivo. Its deletion crucially abolishes assembly of Tim12 in complexes with the other Tims. The N-terminal end contains targeting information and also mediates direct binding of Tim12 to the transmembrane segments of the carrier substrates. These results provide a molecular basis for the concept that the essential role of Tim12 relies on its unique assembly properties that allow this subunit to bridge the soluble and membrane-embedded translocases in the carrier import pathway.     

Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me)

ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36-70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable alpha-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106-715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.     

Origin of the NEFA and Nuc Signal Sequences

The human protein NEFA binds calcium, contains a leucine zipper repeat that does not form a homodimer, and is proposed (along with the homologous Nuc protein) to have a common evolutionary history with an EF-hand ancestor. We have isolated and characterized the N-terminal domain of NEFA that contains a signal sequence inferred from both endoproteinase Asp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to the conserved multiple domains of the mitochondrial carrier family (MCF) proteins. The leader sequence of Nuc is, however, most similar to the signal sequences of membrane and/or secreted proteins (e.g., mouse insulin-like growth factor receptor). We suggest that the divergent NEFA and Nuc N-terminal sequences may have independent origins and that the common high hydrophobicity governs their targeting to the ER. These results provide insights into signal sequence evolution and the multiple origins of protein targeting. Received: 20 February 1997 / Accepted: 28 July 1997     

The N-terminal helix of Bcl-xL targets mitochondria

Anti- and pro-apoptotic Bcl-2 family members regulate the mitochondrial phase of apoptotic cell death. The mitochondrial targeting mechanisms of Bcl-2 family proteins are tightly regulated. Known outer mitochondrial membrane targeting sequences include the C-terminal tail and central helical hairpin. Bcl-x_L also localizes to the inner mitochondrial membrane, but these targeting sequences are unknown. Here we investigate the possibility that the N-terminus of Bcl-x_L also contains mitochondrial targeting information. Amino acid residues 1–28 of Bcl-x_L fused to EGFP are sufficient to target mitochondria. Although positive charges and helical propensity are required for targeting, similar to import sequences the N-terminus is not sufficient for efficient mitochondrial import.     

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.     

Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane

It is assumed that the survival factors Bcl-2 and Bcl-x(L) are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-x(L) is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-x(L) requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.     

The typically mitochondrial DNA-encoded ATP6 subunit of the F1F0-ATPase is encoded by a nuclear gene in Chlamydomonas reinhardtii.

The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.     

Mitochondrial ATP Synthase: A Bioinformatic Approach Reveals New Insights About the Roles of Supernumerary Subunits <Emphasis Type="Italic">g</Emphasis> and A6L

The mitochondrial ATP synthase is a membrane protein complex which couples the proton gradient across the mitochondrial inner membrane to the synthesis of ATP from ADP + P_i. The complex is composed of essential subunits for its motor functions and supernumerary subunits, the roles of which remain to be elucidated. Subunits g and A6L are supernumerary subunits, and the specific roles of these subunits are still matters of debate. To gain insight into the functions of these two subunits, we carried out the alignment and the homolog search of the protein sequences of the subunits and found the following features: Subunit g appears to have isoforms in animals, and the transmembrane domain of the animal subunit g contains a completely conserved acidic residue in the middle of a helix on the conserved side of the transmembrane helix. This finding implicates the conserved acidic residue as important for the function of subunit g. The alignment of A6L protein sequences shows a conserved aromatic residue at the N-terminal domain with which the N-terminal MPQL sequence comprises a unique MPQLX₄Ar motif that can signify the protein A6L. The conserved aromatic residue may also be important for the function of A6L.     

Characterization of the mitochondrial association of hepatitis B virus X protein, HBx

Chronic infection with hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma (HCC). HBx, a protein encoded by HBV is believed to contribute to the development of HCC. HBx was recently shown to associate with mitochondria. In this study, we mapped region(s) of HBx necessary for mitochondrial targeting and showed that a putative transmembrane region (aa 54-70) is required for mitochondrial association. In addition, amino acids in the putative alpha helical regions (aa 75-88 and aa 109-131) seem to aid in the mitochondrial targeting of this protein. We further show that the majority of HBx localizes to the outer mitochondrial membrane based on its sensitivity to trypsin and resistance to alkaline treatment. These studies suggest that the association of HBx with the outer mitochondrial membrane is its intrinsic property. These characterizations define transmembrane and alpha-helical regions of this viral protein as domains of mitochondrial targeting. These studies are further useful in the investigations concerning the physiological significance of the HBx's association with mitochondria and its impact on liver disease pathogenesis.