Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria

3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3- were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3- plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents.     

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C₄ PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in ³²P-labeled inorganic phosphate. In vitro phosphorylation by a Ca²⁺-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme''s velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca²⁺-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C₄ mesophyll protoplasts. These collective data support the hypothesis that this Ca²⁺-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo phosphorylation of a regulatory Ser located in the N-terminal domain of the protein. In vitro phosphorylation by a Ca²⁺-independent, low-molecular-mass (30–39 kD) PEPC-PK modulates PEPC regulation interactively by opposing metabolite effectors (e.g. allosteric activation by Glc-6-P and feedback inhibition by l-malate; Andreo et al., 1987), decreasing significantly the extent of malate inhibition of the leaf enzyme (Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). These metabolites control the rate of phosphorylation of PEPC via an indirect target-protein effect (Wang and Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet, 1997).Several lines of evidence support the view that this protein-Ser/Thr kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The presence and inducible nature of leaf PEPC-PK have been established further in various C₃, C₄, and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent changes in the turnover rate of PEPC-PK itself or another, as yet unknown, protein factor were invoked to account for this observation (Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996). Consistent with this proposal are recent findings about PEPC-PK from leaves of C₃, C₄, and CAM plants that determined activity levels of the enzyme to depend on changes in the level of the corresponding translatable mRNA (Hartwell et al., 1996).Using a cellular approach we previously showed in sorghum (Sorghum bicolor) and hairy crabgrass (Digitaria sanguinalis) that PEPC-PK is up-regulated in C₄ mesophyll cell protoplasts following illumination in the presence of a weak base (NH₄Cl or methylamine; Pierre et al., 1992; Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar to that of the intact, illuminated sorghum (Bakrim et al., 1992) or maize leaf (Echevarría et al., 1990). This light- and weak-base-dependent process via a complex transduction chain is likely to involve sequentially an increase in pHc, inositol trisphosphate-gated Ca²⁺ channels of the tonoplast, an increase in cytosolic Ca²⁺, a Ca²⁺-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK purified on BDA was shown to phosphorylate in vitro both recombinant C₄ PEPC and the root C₃-like isoform, thereby decreasing the enzyme''s malate sensitivity (Pacquit et al., 1993). PEPC from soybean root nodules was phosphorylated in vitro and in vivo by an endogenous PK (Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet, 1997). A Ca²⁺-independent nodule PEPC-PK containing two active polypeptides (32–37 kD) catalyzed the incorporation of phosphate on a Ser residue of the target enzyme and was modulated by photosynthate transported from the shoots (Zhang and Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric (α₂β₂) banana fruit PEPC by a copurifying, Ca²⁺-independent PEPC-PK was shown to occur in vitro (Law and Plaxton, 1997). Although phosphorylation was also detected in vivo and found to concern primarily the α-subunit, PEPC exists mainly in the dephosphorylated form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory phosphorylation in aleurone-endosperm tissue during germination of wheat seeds (Osuna et al., 1996). Here we report on PEPC and the requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a Ca²⁺-independent Ser/Thr PEPC-PK similar to that found in other plant systems studied previously (Chollet et al., 1996); however, the PK was already present in the dry seed and its activity did not require protein synthesis during imbibition.     

Escherichia coli d-Malate Dehydrogenase,a Generalist Enzyme Active in the Leucine Biosynthesis Pathway

The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB⁻ strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution.     

Genes of the Mitochondrial Apoptotic Pathway in Mytilus galloprovincialis

Bivalves play vital roles in marine, brackish, freshwater and terrestrial habitats. In recent years, these ecosystems have become affected through anthropogenic activities. The ecological success of marine bivalves is based on the ability to modify their physiological functions in response to environmental changes. One of the most important mechanisms involved in adaptive responses to environmental and biological stresses is apoptosis, which has been scarcely studied in mollusks, although the final consequence of this process, DNA fragmentation, has been frequently used for pollution monitoring. Environmental stressors induce apoptosis in molluscan cells via an intrinsic pathway. Many of the proteins involved in vertebrate apoptosis have been recognized in model invertebrates; however, this process might not be universally conserved. Mytilus galloprovincialis is presented here as a new model to study the linkage between molecular mechanisms that mediate apoptosis and marine bivalve ecological adaptations. Therefore, it is strictly necessary to identify the key elements involved in bivalve apoptosis. In the present study, six mitochondrial apoptotic-related genes were characterized, and their gene expression profiles following UV irradiation were evaluated. This is the first step for the development of potential biomarkers to assess the biological responses of marine organisms to stress. The results confirmed that apoptosis and, more specifically, the expression of the genes involved in this process can be used to assess the biological responses of marine organisms to stress.     

Fluoroacetic Acid Is a Potent and Specific Inhibitor of Reproduction in the Nematode Caenorhabditis elegans

Fluoroacetic acid is known to lead to inhibition of aconitase and block both the Krebs and glyoxylate cycles. In this study, we discovered it to be a potent and specific inhibitor of reproduction in a bioassay using the nematode Caenorhabditis elegans. Fluoroacetic acid added to the growth medium reduced reproduction in the second generation by 50% at concentrations 3,000 times lower than the concentrations that reduced 24-hour survival by 50%. Four concentrations (2, 4, 8, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations, the survival rates were unaffected, and first-generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. Malonate, which inhibits the Krebs cycle, and itaconate, which inhibits the glyoxylate cycle, were tested individually and in combination. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid. Fluoroacetic acid shows promise as a tool in studies requiring age synchrony.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

AglQ Is a Novel Component of the Haloferax volcanii N-Glycosylation Pathway

N-glycosylation is a post-translational modification performed by members of all three domains of life. Studies on the halophile Haloferax volcanii have offered insight into the archaeal version of this universal protein-processing event. In the present study, AglQ was identified as a novel component of the pathway responsible for the assembly and addition of a pentasaccharide to select Asn residues of Hfx. volcanii glycoproteins, such as the S-layer glycoprotein. In cells deleted of aglQ, both dolichol phosphate, the lipid carrier used in Hfx. volcanii N-glycosylation, and modified S-layer glycoprotein Asn residues only presented the first three pentasaccharide subunits, pointing to a role for AglQ in either preparing the third sugar for attachment of the fourth pentasaccharide subunit or processing the fourth sugar prior to its addition to the lipid-linked trisaccharide. To better define the precise role of AglQ, shown to be a soluble protein, bioinformatics tools were recruited to identify sequence or structural homologs of known function. Site-directed mutagenesis experiments guided by these predictions identified residues important for AglQ function. The results obtained point to AglQ acting as an isomerase in Hfx. volcanii N-glycosylation.     

Oxygen Requirement and Inhibition of C4 Photosynthesis : An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O₂ sensitivity of C₄ photosynthesis was evaluated using a C₄-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C₃-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C₄-cycle-limited mutant showed that atmospheric levels of O₂ (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O₂ partial pressure for photosynthesis was reduced from approximately 5 kPa O₂ to 1 to 2 kPa O₂, becoming similar to that of C₃ plants. Therefore, the higher O₂ requirement for optimal C₄ photosynthesis is specifically associated with the C₄ function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O₂ than in the wild type. When CO₂ fixation by Rubisco is limited, an increase in the CO₂ concentration in bundle-sheath cells via the C₄ cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O₂ while increasing CO₂ leakage and overcycling of the C₄ pathway. These results indicate that in C₄ plants the investment in the C₃ and C₄ cycles must be balanced for maximum efficiency.     

TRIM56 Is an Essential Component of the TLR3 Antiviral Signaling Pathway

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.     

M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.     

Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner Membrane Protein Export Machinery

Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1∼5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.Most mitochondrial proteins are nuclear DNA-coded, and their import into mitochondrial compartments, that is, the mitochondrial outer membrane (MOM),³ mitochondrial inner membrane (MIM), intermembrane space (IMS), and matrix, is mediated by five protein translocation systems: translocase of the outer membrane (TOM complex), sorting and assembly machinery of MOM (SAM/TOB), translocases of the inner membrane (TIM23 complex and TIM22 complex), and a fifth system in the MIM that mediates integration of proteins from the matrix into the MIM (1, 2). The last system, which has been analyzed in detail in yeast, requires a membrane potential across the MIM and matrix ATP and mediates MIM integration of the mtDNA-encoded proteins as well as the integration of certain nuclear DNA-encoded proteins considered to be of bacterial origin, such as cytochrome c oxidase subunit II, F1Fo-ATPase subunit 9, and Oxa1 (3–5). Translocation efficiency is affected by the charge difference across the transmembrane (TM) in accordance with the positive-inside rule (5). Furthermore, the matrix-exposed C-terminal segment of Oxa1 is essential for binding mitochondrial ribosomes during cotranslational integration of mtDNA-encoded proteins (6, 7). Recent reports further demonstrated that the MIM protein Mba1, as a ribosome receptor, cooperates with the C-terminal ribosome binding segment of Oxa1 (8). The machinery and the underlying mechanisms of MIM insertion from the matrix must be further analyzed.Oxa1 protein, originally identified in yeast, is a component of the matrix-to-MIM export system conserved from prokaryote to eukaryote and is involved in Oxa1 biogenesis (9–14). YidC, a bacterial homologue of Oxa1, is involved in the biogenesis of inner membrane proteins in a Sec-dependent or Sec-independent manner (15, 16). In yeast, IMS export from the matrix of the Oxa1 N-terminal segment emerging from the Tim23 channel requires a membrane potential (4, 17), and the export is compromised in mitochondria isolated from a temperature-sensitive Oxa1-expressing strain at a non-permissive temperature (12). Herrmann and Bonnefoy (18) reported that Oxa1 protein functions in the export of a single hydrophilic loop region that was artificially produced by ligating the C-terminal region of cytochrome b with cytochrome c oxidase subunit II and placed between TM segments. Direct interaction of Oxa1 with an immature subunit in complex V was observed during its biogenesis (19). So far, these studies have only been performed in yeast, and no information is available on the mechanism of topogenesis in mammals with regard to how Oxa1 is involved in the export of multiple regions in a protein molecule. Our in vivo study revealed that the correct topogenesis of Oxa1 in the MIM proceeds as a result of the cooperation of all five TMs and that the cooperation of TM1 and the species-conserved six-residue segment (GLPWWG) in the N-terminal flanking region is essential for export from the matrix of both the N-terminal segment and hydrophilic L2 into the IMS.