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• W2017455861 abstract "Article1 June 1999free access Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein Yasuo Nemoto Yasuo Nemoto Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Pietro De Camilli Corresponding Author Pietro De Camilli Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Yasuo Nemoto Yasuo Nemoto Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Pietro De Camilli Corresponding Author Pietro De Camilli Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510 USA Search for more papers by this author Author Information Yasuo Nemoto1 and Pietro De Camilli 1 1Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06510 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:2991-3006https://doi.org/10.1093/emboj/18.11.2991 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Synaptojanin 1 is an inositol 5′-phosphatase highly enriched in nerve terminals with a putative role in recycling of synaptic vesicles. We have previously described synaptojanin 2, which is more broadly expressed as multiple alternatively spliced forms. Here we have identified and characterized a novel mitochondrial outer membrane protein, OMP25, with a single PDZ domain that specifically binds to a unique motif in the C-terminus of synaptojanin 2A. This motif is encoded by the exon sequence specific to synaptojanin 2A. OMP25 mRNA is widely expressed in rat tissues. OMP25 is localized to the mitochondrial outer membrane via the C-terminal transmembrane region, with the PDZ domain facing the cytoplasm. Overexpression of OMP25 results in perinuclear clustering of mitochondria in transfected cells. This effect is mimicked by enforced expression of synaptojanin 2A on the mitochondrial outer membrane, but not by the synaptojanin 2A mutants lacking the inositol 5′-phosphatase domain. Our findings provide evidence that OMP25 mediates recruitment of synaptojanin 2A to mitochondria and that modulation of inositol phospholipids by synaptojanin 2A may play a role in maintenance of the intracellular distribution of mitochondria. Introduction Growing evidence has indicated that inositol metabolism plays pivotal roles in membrane trafficking processes within cells, in addition to its well established role in intracellular signaling pathways. An emerging concept is that various phosphoinositide stereoisomers, with phosphate groups at different positions of the inositol ring, are recognized by specific proteins that are involved in generation of membrane vesicles and in their docking to acceptor compartments. Thus, changes in phosphoinositide levels in a spatially and temporally regulated manner are means by which membrane trafficking events are controlled. These mechanisms seem to underlie the requirements for several enzymes and proteins responsible for synthesis, intracellular transport and breakdown of distinct phosphoinositides in specific membrane trafficking steps (Roth and Sternweis, 1997; Martin, 1998, and references therein). We have recently identified a neuronal inositol polyphosphate 5′-phosphatase, synaptojanin 1 (McPherson et al., 1994a, 1996) and a more broadly expressed form, synaptojanin 2 (Nemoto et al., 1997). Synaptojanins dephosphorylate inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate at the D-5 position of the inositol ring in vitro (McPherson et al., 1996; Nemoto et al., 1997; Woscholski et al., 1997) and define a subfamily of inositol 5′-phosphatases with a unique three-domain structure: an N-terminal domain homologous to Saccharomyces cerevisiae Sac1p (Cleves et al., 1989; Novick et al., 1989), a central inositol 5′-phosphatase domain and a C-terminal proline-rich domain. Synaptojanin 1 and dynamin 1 are highly enriched in nerve terminals, where they are localized on coated endocytic intermediates (McPherson et al., 1994b; Haffner et al., 1997). Both proteins undergo dephosphorylation in response to nerve terminal depolarization (Robinson et al., 1993; McPherson et al., 1994b; Bauerfeind et al., 1997), and it has been shown recently that such depolarization-induced dephosphorylation promotes the assembly of several proteins for compensatory endocytosis in nerve terminals (Slepnev et al., 1998). Synaptojanin 1 and dynamin 1 specifically interact with the SH3 domains of Grb2 (Gout et al., 1993; McPherson et al., 1994a), amphiphysin 1 and 2 (David et al., 1996; Micheva et al., 1997; Ramjaun et al., 1997), members of the SH3p4/8/13 protein family (endophilins) (de Heuvel et al., 1997; Ringstad et al., 1997), intersectin (Yamabhai et al., 1998) and syndapin I (Qualmann et al., 1999). These properties suggest that synaptojanin 1, together with dynamin 1, functions in the retrieval of the synaptic vesicle membrane by clathrin-coated vesicles. Inositol phospholipids have been reported to bind to several proteins implicated in this process with high affinity, and in some cases modulate their biochemical properties. Examples include the α-subunit of clathrin assembly protein complex AP2, the neuron-specific clathrin assembly protein AP180, the C2B domain of synaptotagmin and the pleckstrin homology domain of dynamin 1 (reviewed in Cremona and De Camilli, 1997). Furthermore, we and others have recently shown that deletion of yeast synaptojanin-like genes causes a severe defect in receptor-mediated and fluid phase endocytosis (Singer-Krüger et al., 1998; Stolz et al., 1998). These results, together with those of Luo and Chang (1997), provided the first direct evidence for a role for synaptojanins in endocytic membrane trafficking in S.cerevisiae. Synaptojanin 2 undergoes extensive alternative splicing in the C-terminal proline-rich region to generate multiple isoforms (Khvotchev and Südhof, 1998; Seet et al., 1998). Our initial characterization of synaptojanin 2 (now referred to as synaptojanin 2A) showed that synaptojanin 2A is associated predominantly with the particulate fraction, while synaptojanin 1 is localized mainly in the soluble fraction in transfected Chinese hamster ovary (CHO) cells, and that the different protein–protein interactions in their proline-rich region are likely to direct synaptojanin 1 and 2A to different subcellular compartments (Nemoto et al., 1997). To explore the biochemical basis of the previous findings, in this study we performed a yeast two-hybrid screening for synaptojanin 2A-specific interacting proteins and we have identified a novel mitochondrial outer membrane protein with a single PDZ domain, OMP25. We show that the PDZ domain of OMP25 binds to the unique motif in the C-terminal sequence specific to synaptojanin 2A among several alternatively spliced forms of synaptojanin 2. By this interaction, OMP25 mediates the recruitment of synaptojanin 2A to mitochondria, where it causes an alteration of morphology of the mitochondrial network. OMP25 represents the first identified component in mammalian mitochondria that appears to play a role in the proper distribution of mitochondria. Our observations implicate the specific pools of inositol phospholipids in regulation of the dynamics of intracellular organelles and extend the roles of inositol metabolism in cell physiology. Results Identification of OMP25, a synaptojanin 2A-interacting transmembrane protein with a single PDZ domain We have searched for proteins that specifically interact with the proline-rich region of synaptojanin 2A and not with that of synaptojanin 1 by yeast two-hybrid screening. Thirty-six clones were obtained and they were independent isolates of four overlapping cDNAs encoding a portion of a novel protein. We refer to this protein as OMP25 (for outer membrane protein of 25 kDa). A full-length cDNA for OMP25 was isolated by screening of rat brain cDNA libraries and a PCR-mediated procedure. The composite cDNA sequence for rat OMP25 is 5.2 kb long (Figure 1A). An extraordinarily long 3′-untranslated region (3′-UTR) is AU-rich and contains multiple AUUUA repeats (underlined in Figure 1A), which are implicated in the instability of mRNAs and often found in transiently expressed mRNAs encoding cytokines and proto-oncogene products (Treisman, 1985; Shaw and Kamen, 1986), suggesting that expression of OMP25 may be dynamically regulated. An open reading frame (ORF) of 621 bases encodes a protein of 206 amino acids with a predicted Mr of 22 564 and pI of 9.46. The putative initiator codon at nucleotides 456–458 is preceded by an in-frame termination codon in the 5′-UTR at nucleotides 312–314. Sequence analysis revealed that OMP25 has a single PDZ domain (amino acids 73–160) that is most similar to the first and second PDZ domains of DLG/SAP97 (Lue et al., 1994; Müller et al., 1995) and PSD-95/SAP90 (Cho et al., 1992; Kistner et al., 1993) (Figure 1B). The region preceding the PDZ domain, especially the N-terminal portion (amino acids 1–50), is rich in basic amino acid residues (pI = 11. 43) and predicted to adopt the amphipathic α-helix structure (data not shown). Near the C-terminus, OMP25 has a 23 amino acid stretch of hydrophobic residues that is predicted to form a membrane-spanning region, as indicated by the hydrophilicity profile (Figure 1C). Searches for OMP25-related sequences showed the presence of highly homologous expressed sequence tags (ESTs) in a variety of human and mouse tissues, which seem to be derived from the OMP25 homologs (Figure 1D). Figure 1.Primary structure of rat OMP25. (A) Nucleotide and deduced amino acid sequence of rat OMP25 cDNA. The composite nucleotide sequence of rat OMP25 cDNA is shown. The predicted amino acid sequence is shown below in the three letter code. An in-frame termination codon in the 5′-UTR and potential mRNA-destabilizing elements in the 3′-UTRs are underlined. A PDZ domain and a transmembrane region in OMP25 are indicated by bold letters and italics, respectively. Nucleotides are numbered on the left and amino acid residues are numbered on the right. The sequence data are available from DDBJ/EMBL/GenBank under accession No. AF107295>. (B) The PDZ domains of rat OMP25 (amino acids 73–160), rat DLG/SAP97 (amino acids 223–310, 318–404 and 464–545) and rat PSD-95/SAP90 (amino acids 64–151, 160–246 and 312–393) are aligned by the Clustal method (Higgins and Sharp, 1988). The residues corresponding to the secondary structural elements identified in the third PDZ domain of PSD-95 are indicated according to Doyle et al. (1996). (C) Schematic representation of the domain structure and the Kyte–Doolittle hydrophilicity profile of OMP25 (Kyte and Doolittle, 1982). OMP25 has a putative transmembrane segment near the C-terminus (amino acids 178–200). (D) Alignment of rat OMP25 and the putative mouse and human homologs. The partial amino acid sequences of putative mouse and human OMP25 are derived from the composite sequences of mouse ESTs AA254858, AA221650, AI317677 and AU044272 and human ESTs AA075102, AA369660, AI309021 and W63718, respectively. The amino acid residues shared by all the homologs are shown by black boxes. The residues corresponding to the transmembrane segments are underlined. Missing amino acids are indicated by dashes. Amino acid residues of rat OMP25 are numbered on the right. Download figure Download PowerPoint The C-terminal 16 amino acid residues from the synaptojanin 2A-specific exon sequence are sufficient for selective binding to OMP25 Inspection of the synaptojanin 2A sequence revealed that the C-terminal region of synaptojanin 2A conforms to a tS/TXV motif (see Discussion). We prepared GST fusions of the PDZ domain of OMP25 (GST–OMP25PDZ) and of all three PDZ domains of DLG (GST–DLG3PDZ) as a control to study the biochemical interaction between synaptojanin 2A and OMP25. We first tested whether the specific interaction is mediated by the PDZ domain of OMP25 by blot overlay of the extracts from cultured cells transiently expressing FLAG-tagged synaptojanin 1 or 2A. As shown in Figure 2A, while the extracts contained comparable amounts of recombinant synaptojanin 1 and 2A as judged by immunoblotting with anti-FLAG antibody, GST–OMP25PDZ bound to synaptojanin 2A, but not to synaptojanin 1. By contrast, GST–DLG3PDZ bound to neither synaptojanin 1 nor synaptojanin 2A under the conditions used. Furthermore, a 140 kDa protein was affinity purified from rat brain extract selectively by GST–OMP25PDZ and the protein was shown to be synaptojanin 2A by immunoblotting, illustrating the highly specific interaction between synaptojanin 2A and the PDZ domain of OMP25 (Figure 2B). Figure 2.Specific binding of the C-terminal region of synaptojanin 2A to the PDZ domain of OMP25. (A) Blot overlay of the recombinant synaptojanins by GST–PDZ domain fusion proteins. Lysates from COS-7 cells expressing either FLAG-tagged synaptojanin 1 (FLAG-SJ1) or synaptojanin 2A (FLAG-SJ2A) and mock-transfected cells (Mock) were probed by anti-FLAG antibody or the indicated GST–PDZ domain fusion protein. (B) Affinity purification of synaptojanin 2A from rat brain by the PDZ domain of OMP25. A Triton X-100 extract of rat brain was affinity purified onto the indicated GST fusion proteins. Bound proteins were released by Laemmli sample buffer and either stained by Coomassie Brilliant Blue (left panel) or analyzed by immunoblotting with anti-synaptojanin 2A antibody (right panel). The 145 and 100 kDa proteins purified by the SH3 domain of SH3p4 (endophilin 1) have been identified as synaptojanin 1 (145 kDa form) and dynamin 1, respectively (Ringstad et al., 1997). In the eluate from GST–DLG3PDZ, a 60 kDa protein is the fusion protein. The 160 kDa protein was not identified in this study. (C) MBP fusion proteins of the four overlapping segments of the synaptojanin 2A proline-rich region (numbered 1–4, from the N- to the C-terminus) were probed by anti-MBP antibody or the indicated GST–PDZ domain fusion protein. (D) Binding of GST–PDZ fusion proteins to a 16 amino acid C-terminal peptide of synaptojanin 2A. GST–PDZ fusion proteins were incubated with a bead-immobilized peptide corresponding to the C-terminal 16 amino acid residues of rat synaptojanin 2A, or with a control peptide corresponding to the N-terminal 18 amino acid residues of rat synaptojanin 2A. Bound fusion proteins were eluted with Laemmli sample buffer and detected by immunoblotting with anti-GST antibody. (E) Dose–response curves of binding of GST–PDZ domain fusion proteins to the synaptojanin 2A C-terminal peptide in an ELISA assay. Download figure Download PowerPoint To define the region of synaptojanin 2A required for the association with OMP25, we divided the proline-rich region of synaptojanin 2A into four overlapping segments. Each segment was expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein or in S.cerevisiae as a LexA fusion protein, and the binding to OMP25 was tested by blot overlay and yeast two-hybrid assays. In the blot overlay, the most C-terminal segment bound to the PDZ domain of OMP25, whereas other segments showed no detectable binding (Figure 2C). Again the PDZ domains of DLG failed to bind to any segments of the proline-rich region of synaptojanin 2A. The results were corroborated by the yeast two-hybrid assay (data not shown). To narrow down further the region of synaptojanin 2A mediating the specific binding to the PDZ domain of OMP25, a synthetic peptide corresponding to the last 16 C-terminal amino acid residues of synaptojanin 2A, which is embedded in the synaptojanin 2A-specific exon sequence among various alternatively spliced forms of synaptojanin 2 (Khvotchev and Südhof, 1998; Seet et al., 1998), was coupled to gel beads via the N-terminal end. The beads were incubated with the GST–PDZ domain fusion proteins and the bound protein was eluted and detected by immunoblotting with anti-GST antibody. As a control, a peptide corresponding to the N-terminal end of synaptojanin 2A was coupled to the gel and the binding to the GST–PDZ domain fusion proteins was tested similarly. The C-terminal 16 amino acid peptide was capable of binding to the GST–OMP25PDZ domain, while it did not bind to GST–DLG3PDZ. No binding between the N-terminal peptide and the GST–PDZ domains was detected (Figure 2D). The relative binding affinity of the PDZ domains in OMP25 and DLG for the C-terminal peptide of synaptojanin 2A was measured by an enzyme-linked immunosorbent assay (ELISA) using a series of concentrations of the GST fusion proteins (Figure 2E). Analysis of the dose–response curve gave an EC50 value of 21 nM. This value is comparable with the apparent EC50 between the second PDZ domain of SAP102 and the C-terminal peptide of the N-methyl-D-aspartate (NMDA) receptor measured by a similar method (Müller et al., 1996), further suggesting that this interaction may be physiologically relevant. The assay suggested that GST–DLG3PDZ has an EC50 >10 μM, implying that the PDZ domain of OMP25 has at least 100-fold higher affinity for the synaptojanin 2A C-terminal peptide than the PDZ domains in DLG. Binding of the PDZ domains to the synaptojanin 2A N-terminal peptide was negligible (data not shown). Collectively, these results indicate that the interaction of synaptojanin 2A with OMP25 is mediated by the 2A-specific exon sequence at its most C-terminal end among various alternatively spliced forms of synaptojanin 2. The PDZ domain of OMP25 recognizes a unique C-terminal motif of synaptojanin 2A Our in vitro protein binding experiments have shown that the C-terminal sequence of synaptojanin 2A binds to the PDZ domain of OMP25, but not to the PDZ domains of rat DLG, despite their close similarity. We therefore addressed the structural requirement for the specific binding of synaptojanin 2A to the PDZ domain of OMP25 by mutagenesis analysis. A series of C-terminal mutant proteins in which the residues at different positions of the five most C-terminal amino acid residues of synaptojanin 2A (-SGSSV) were replaced with alanine residues were expressed and their binding to the PDZ domain of OMP25 was analyzed by blot overlay and yeast two-hybrid assays (Figure 3A and B). Replacement of the serine residue in position −2 or the C-terminal valine residue abolished the binding to the PDZ domain, whereas replacement of the serine residues in position −1 or −4 did not affect the binding, indicating that the residues in the C-terminus and position −2 are essential for binding to the OMP25 PDZ domain, in agreement with studies on other PDZ domain-mediated interactions. Figure 3.Alanine replacement mutagenesis of the C-terminal region of synaptojanin 2A. (A) Blot overlay. The five most C-terminal amino acid residues of synaptojanin 2A (-SGSSV) in segment IV of the proline-rich region were replaced by alanine residues and expressed as MBP fusion proteins. The wild-type and mutant proteins were probed by anti-MBP antibody or GST–OMP25PDZ. (B) Yeast two-hybrid assay. The wild-type and the segment IV mutants were expressed as the LexA fusion protein in AMR70. The strains were mated with L40 expressing the GAL4 activation domain fusion protein of the PDZ domain of OMP25. The activity of β-galactosidase in the resultant dipolid strains was measured in triplicate and expressed as the mean values ± SD in Miller units. Download figure Download PowerPoint Somewhat unexpectedly, when the glycine residue in position −3 was replaced by an alanine residue, the binding of the mutant protein to the PDZ domain of OMP25 was diminished significantly, suggesting that this residue is also important for the effective binding of the ligand to the OMP25 PDZ domain. A recent study on the binding specificities of several PDZ domains by the oriented peptide library technique showed that the PDZ domains of mouse DLG bind preferentially to peptides with an ES/TXV/I motif, with a glutamate residue at position −3 (Songyang et al., 1997). The difference in the preferred recognition motifs at the −3 peptide position may account in part for the observed specific interaction between the C-terminus of synaptojanin 2A and OMP25. Remarkably, the C-terminus of human synaptojanin 2A deduced from an EST (accession No. AI014555) ends in -SGSSV, supporting the importance of the glycine residue at position −3. OMP25 mRNA is widely distributed in rat tissues We performed a series of Northern blot experiments to determine the tissue distribution of OMP25 mRNA and to verify the authenticity of the OMP25 cDNA. We used three probes derived from the coding region and the distal and middle parts of the 3′-UTR of OMP25 cDNA (Figure 4). The probe corresponding to the PDZ domain of OMP25 detected RNA species of 6.0, 3.6, 3.4 and 1.0 kb with varying relative abundance in all the adult rat tissues tested (Figure 4A). A probe from the distal 3′-UTR detected only the largest 6 kb transcript (Figure 4C), While a probe from the middle 3′-UTR hybridized to the largest 6 kb and the middle 3.6 and 3.4 kb bands and not to the smallest 1 kb band (Figure 4B). These results indicate that the isolated OMP25 cDNA corresponds to the largest 6 kb transcript, and other smaller transcripts arise by alternative splicing or alternative usage of polyadenylation sites within the 3′-UTR. Thus, OMP25 mRNA is widely expressed in rat tissues as multiple transcripts. Figure 4.Tissue distribution of rat OMP25 mRNA. OMP25 transcripts in various adult rat tissues were detected by Northern blot analysis with the probes derived from the open reading frame (A), the middle (B) and the distal (C) parts of the 3′-UTR of the isolated OMP25 cDNA, as indicated at the top. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. RNA size markers are indicated in kb on the left. Download figure Download PowerPoint OMP25 is localized to mitochondria via the C-terminal transmembrane region and its overexpression induces clustering of mitochondria To investigate the possible biological function of OMP25, we expressed OMP25 with a hemagglutinin (HA) epitope tag at the N-terminus in CHO cells and examined its intracellular localization by indirect immunofluorescence. Overexpressed OMP25 was localized on punctate and rod-like structures. An increased level of expression correlated with coalescence of these structures in the perinuclear region, and in highly overexpressing cells they formed one or a few masses. The structures were found to be coincident with mitochondria as detected by antibody against cytochrome c reductase (complex III) or cytochrome c oxidase (complex IV) (Figure 5). The structures were also recognized by MitoTracker, a mitochondria-specific fluorescent dye whose uptake depends on the mitochondrial membrane potential (Poot et al., 1996), confirming the mitochondrial localization of OMP25 and the preservation of the mitochondrial membrane potential despite the grossly altered distribution and morphology. OMP25 in the transfected cells did not co-localize with markers for other organelles tested including the endoplasmic reticulum, the Golgi and endosomes (data not shown). Figure 5.Co-localization of HA-tagged OMP25 with mitochondria in CHO cells. Immunofluorescence of CHO cells transfected with the HA epitope-tagged OMP25 construct. The cells were double-stained with mouse monoclonal anti-HA antibody (a and c) and rabbit polyclonal antibody against cytochrome c oxidase (b and d). Scale bars correspond to 10 μm. Note the perinuclear clustering of mitochondria in transfected cells (a and b) which starts from bundling of mitochondria in cells expressing low levels of OMP25 (c and d). Download figure Download PowerPoint To determine the region required for targeting of OMP25 to mitochondria, we expressed epitope-tagged truncated proteins and examined their localization by indirect immunofluorescence. The protein lacking the N-terminal basic region (OMP25ΔN) exhibited a distribution indistinguishable from that of intact OMP25 and induced mitochondrial clustering (Figure 6A, a and b). In contrast, the protein without the transmembrane region (OMP25ΔC) was localized diffusely throughout the cytoplasm and did not perturb mitochondrial distribution (Figure 6A, c and d). Consistent with the immunofluorescence localization, subcellular fractionation of the transfected cells showed that intact OMP25 and OMP25ΔN were associated with the particulate fraction, whereas OMP25ΔC was recovered in the soluble fraction (Figure 6B). These results indicate that the C-terminal transmembrane region of OMP25 is essential for localization of OMP25 to mitochondria, while the N-terminal region is dispensable, although the positively charged and amphipathic helical nature of the region bears some resemblance to mitochondrial pre-sequences (Roise and Schatz, 1988). Figure 6.The C-terminal transmembrane region is essential for mitochondrial localization of OMP25. (A) Immunofluorescence of CHO cells transfected with the HA epitope-tagged construct for OMP25 lacking either its N-terminal region (a and b) or its C-terminal transmembrane region (c and d). The cells were double-stained with mouse monoclonal anti-HA antibody (a and c) and rabbit polyclonal antibody against cytochrome c oxidase (b and d). The scale bar corresponds to 10 μm. (B) CHO cells were transfected as above and the total homogenates (T) were fractionated into the soluble (S) and the particulate fractions (P). The localization of OMP25 and its truncations was detected by immunoblotting with anti-HA antibody. (C) CHO cells were transfected with the construct for GFP fused to the C-terminal transmembrane region of OMP25. The cells were incubated with MitoTracker and then fixed. Fluorescence of GFP (a) and MitoTracker (b) was detected by confocal laser scanning microscopy. The scale bar corresponds to 10 μm. (D) Schematic representation of the structure of OMP25 and its variants and summary of the localization. Download figure Download PowerPoint To test further whether the C-terminal transmembrane region is sufficient for mitochondrial targeting, we expressed green fluorescent protein (GFP) in fusion with the transmembrane region of OMP25. As shown in Figure 6C, the GFP fusion protein co-localizes with mitochondria and does not affect the mitochondrial distribution, demonstrating that the transmembrane region of OMP25 contains all the essential information for mitochondrial targeting and has the ability to direct a heterologous protein to mitochondria. OMP25 is an integral protein in the mitochondrial outer membrane with the PDZ domain facing the cytoplasm The transient expression study of epitope-tagged OMP25 constructs indicates that OMP25 is localized to mitochondria via its C-terminal transmembrane region. The presence of an integral membrane protein with a PDZ domain in an intracellular organelle is unprecedented. To rule out the possibility that the addition of a negatively charged epitope sequence might affect the observed localization of OMP25, we tested the mitochondrial localization of OMP25 by independent approaches. We probed the localization of anti-OMP25 immunoreactivity in rat liver mitochondria (Figure 7A). An antibody against the N-terminal peptide from OMP25 recognized a 25 kDa band in the mitochondrial fraction. The immunoreactive protein could not be extracted by alkali carbonate treatment (Fujiki et al., 1982) and was degraded by proteinase K treatment, indicating a mitochondrial membrane localization with the bulk of the protein exposed to the cytosol. Figure 7.OMP25 is an integral protein in the mitochondrial outer membrane. (A) Localization of OMP25 immunoreactivity in the rat liver mitochondria fraction. Left panel: mitochondria and post-mitochondrial supernatant (Post-Mito Sup) were prepared from rat liver homogenate. Each fraction (50 μg protein/lane) was subjected to immunoblotting with an antibody against the N-terminal peptide of OMP25 or an antibody against cytochrome c oxidase subunit IV (COXIV). Right panel: the isolated mitochondria were treated either with 0.1 M Na2CO3, pH 11.5 or 0.1 mg/ml proteinase K, as indicated, and subjected to immunoblotting. (B) In vitro import of OMP25 into the isolated mitochondria. Top panel: OMP25 was synthesized in reticulocyte lysate in the presence of [35S]methionine (lane 1, 10% of the radiolabeled precursor protein) and incubated with the isolated rat liver mitochondria in the presence (lanes 5–7) or absence (lanes 2–4) of 1.0 μM CCCP. After the import, mitochondria were recovered by centrifugation (lanes 2 and 5). Portions were either resuspended in 0.1 M Na2CO3, pH 11.5, and the alkali-inextractable fractions obtained (lanes 3 and 6), or treated with 0.1 mg/ml proteinase K (lanes 4 and 7). Middle panel: radiolabeled human pre-OCT was synthesized and processed in the same way. Bottom panel: the samples were analyzed by immunoblotting with an antibody against COXIV. Download figure Download PowerPoint Radiolabeled OMP25 was synthesized in vitro by usin" @default.
• W2017455861 created "2016-06-24" @default.
• W2017455861 creator A5037459294 @default.
• W2017455861 creator A5082148877 @default.
• W2017455861 date "1999-06-01" @default.
• W2017455861 modified "2023-10-11" @default.
• W2017455861 title "Recruitment of an alternatively spliced form of synaptojanin 2to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein" @default.
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