FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079676584> ?p ?o ?g. }

• W2079676584 endingPage "27788" @default.
• W2079676584 startingPage "27781" @default.
• W2079676584 abstract "The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, associated with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation (Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M., Shaw, J. M., and Nunnari, J. (2000) J. Cell Biol. 151, 341–352; Wong, E. D., Wagner, J. A., Scott, S. V., Okreglak, V., Holewinske, T. J., Cassidy-Stone, A., and Nunnari, J. (2003) J. Cell Biol. 160, 303–311; Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in press). Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, we have characterized these isoforms. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphology. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Δpcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Δmgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphology and maintenance of mitochondrial DNA. The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, associated with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation (Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M., Shaw, J. M., and Nunnari, J. (2000) J. Cell Biol. 151, 341–352; Wong, E. D., Wagner, J. A., Scott, S. V., Okreglak, V., Holewinske, T. J., Cassidy-Stone, A., and Nunnari, J. (2003) J. Cell Biol. 160, 303–311; Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in press). Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, we have characterized these isoforms. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphology. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Δpcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Δmgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphology and maintenance of mitochondrial DNA. Mitochondria are dynamic structures. Their overall morphology and the shape of the inner membrane are highly variable (5Reichert A.S. Neupert W. Biochim. Biophys. Acta. 2002; 1592: 41-49Crossref PubMed Scopus (101) Google Scholar). This is exemplified by a wide variety of mitochondrial ultrastructures observed in different organisms and tissues and by the dependence of these structures on the metabolic and genetic state of the cell. Mitochondrial dynamics has recently gained increasing attention, and quite a number of proteins affecting this structural diversity have been identified. Still, our understanding of the underlying molecular events is rather incomplete. Fusion and fission of mitochondrial membranes are processes that have to be balanced in order to maintain mitochondrial morphology (6Sesaki H. Jensen R.E. J. Cell Biol. 1999; 147: 699-706Crossref PubMed Scopus (436) Google Scholar). For example, when fusion of mitochondria is abolished, fission can still continue, and consequently mitochondria get progressively fragmented (reviewed in Ref. 7Westermann B. EMBO Rep. 2002; 3: 527-531Crossref PubMed Scopus (121) Google Scholar). The roles of a number of proteins such as the GTPase Fzo1 (8Fritz S. Rapaport D. Klanner E. Neupert W. Westermann B. J. Cell Biol. 2001; 152: 683-692Crossref PubMed Scopus (121) Google Scholar) and the dynamin-like GTPase Dnm1 (9Bleazard W. McCaffery J.M. King E.J. Bale S. Mozdy A. Tieu Q. Nunnari J. Shaw J.M. Nat. Cell. Biol. 1999; 1: 298-304Crossref PubMed Scopus (598) Google Scholar) in fusion and fission are known. Less clear is the role of the mitochondrial dynamin-like protein Mgm1, which is another component essential for maintaining mitochondrial morphology in Saccharomyces cerevisiae. Deletion of the corresponding gene in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA (10Guan K. Farh L. Marshall T.K. Deschenes R.J. Curr. Genet. 1993; 24: 141-148Crossref PubMed Scopus (116) Google Scholar, 11Jones B.A. Fangman W.L. Genes Dev. 1992; 6: 380-389Crossref PubMed Scopus (193) Google Scholar, 12Shepard K.A. Yaffe M.P. J. Cell Biol. 1999; 144: 711-720Crossref PubMed Scopus (147) Google Scholar). Mutations in the orthologous gene in humans, OPA1, are associated with optic atrophy type I (reviewed in Ref. 13Delettre C. Lenaers G. Pelloquin L. Belenguer P. Hamel C.P. Mol. Genet. Metab. 2002; 75: 97-107Crossref PubMed Scopus (147) Google Scholar). Dynamins are a family of large GTPases involved in membrane fission during endocytosis (reviewed in Ref. 14Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar). However, the physiological roles of the mitochondrial dynamin-like proteins Mgm1 and OPA1 are not understood very well. Several hypotheses have been put forward that are mainly based on the homology to dynamins, on the mitochondrial localization, and the fragmentation of mitochondria in strains in which Mgm1 is mutated or deleted (10Guan K. Farh L. Marshall T.K. Deschenes R.J. Curr. Genet. 1993; 24: 141-148Crossref PubMed Scopus (116) Google Scholar, 11Jones B.A. Fangman W.L. Genes Dev. 1992; 6: 380-389Crossref PubMed Scopus (193) Google Scholar, 12Shepard K.A. Yaffe M.P. J. Cell Biol. 1999; 144: 711-720Crossref PubMed Scopus (147) Google Scholar). According to one hypothesis, Mgm1 modulates the morphology of the inner membrane and therefore is thought to be involved in cristae formation and/or inner membrane fission events (2Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. Nunnari J. J. Cell Biol. 2000; 151: 341-352Crossref PubMed Scopus (269) Google Scholar). Down-regulation of OPA1 in HeLa cells by small interfering RNA was reported to alter cristae morphology (15Olichon A. Baricault L. Gas N. Guillou E. Valette A. Belenguer P. Lenaers G. J. Biol. Chem. 2003; 278: 7743-7746Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar). On the other hand, two recent reports indicate an important role for Mgm1 in the fusion of mitochondria (3Wong E.D. Wagner J.A. Scott S.V. Okreglak V. Holewinske T.J. Cassidy-Stone A. Nunnari J. J. Cell Biol. 2003; 160: 303-311Crossref PubMed Scopus (196) Google Scholar, 4Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in pressGoogle Scholar). Contradicting views exist about the exact submitochondrial location of Mgm1. It is still a matter of debate whether Mgm1 is located in the outer membrane (12Shepard K.A. Yaffe M.P. J. Cell Biol. 1999; 144: 711-720Crossref PubMed Scopus (147) Google Scholar) or in the intermembrane space of mitochondria (2Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. Nunnari J. J. Cell Biol. 2000; 151: 341-352Crossref PubMed Scopus (269) Google Scholar, 3Wong E.D. Wagner J.A. Scott S.V. Okreglak V. Holewinske T.J. Cassidy-Stone A. Nunnari J. J. Cell Biol. 2003; 160: 303-311Crossref PubMed Scopus (196) Google Scholar, 4Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in pressGoogle Scholar) and whether its Schizosaccharomyces pombe ortholog, Msp1, resides in the matrix (16Pelloquin L. Belenguer P. Menon Y. Gas N. Ducommun B. J. Cell Sci. 1999; 112: 4151-4161PubMed Google Scholar). Mgm1 and its orthologs from all species investigated so far exist in at least two isoforms (2Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. Nunnari J. J. Cell Biol. 2000; 151: 341-352Crossref PubMed Scopus (269) Google Scholar, 12Shepard K.A. Yaffe M.P. J. Cell Biol. 1999; 144: 711-720Crossref PubMed Scopus (147) Google Scholar, 17Olichon A. Emorine L.J. Descoins E. Pelloquin L. Brichese L. Gas N. Guillou E. Delettre C. Valette A. Hamel C.P. Ducommun B. Lenaers G. Belenguer P. FEBS Lett. 2002; 523: 171-176Crossref PubMed Scopus (335) Google Scholar, 18Misaka T. Miyashita T. Kubo Y. J. Biol. Chem. 2002; 277: 15834-15842Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 19Pelloquin L. Belenguer P. Menon Y. Ducommun B. Biochem. Biophys. Res. Commun. 1998; 251: 720-726Crossref PubMed Scopus (66) Google Scholar). Thus, it is crucial to clarify the identity of these isoforms and how they are generated. Furthermore, it is important to unravel the function of each of these isoforms. Are they both necessary for wild type mitochondrial morphology and maintenance of mitochondrial DNA, or do they serve independent functions? Here we present data that provide new insights into the biogenesis of Mgm1 and the role of its two isoforms for wild type-like mitochondrial morphology and maintenance of mtDNA 1The abbreviations used are: mtDNA, mitochondrial DNA; DAPI, 4′,6-diamidino-2-phenylindole; MPP, mitochondrial processing peptidase; s-Mgm1, short isoform of Mgm1; l-Mgm1, large isoform of Mgm1. in yeast. Plasmids and Yeast Strains—Mgm1-(1–228) was amplified from genomic yeast DNA (W303) using the primer 5′-CCC CGA ATT CGA GCT CGC CAT GAG TAA TTC TAC TTC ATT AAG G-3′ and the reverse primer 5′-CCC CGG ATC CAT GTG CAG AAG AAG AGT CC-3′. The PCR product was digested with EcoRI and BamHI and cloned in frame into pGEM4 (Invitrogen) containing mouse dihydrofolate reductase between the BamHI and HindIII restriction sites to yield a dihydrofolate reductase fusion protein. Mgm1-(1–427) was amplified using 5′-CCC CGC GGC CGC TCT AGA TCA GAG AGG ATA TTT TTT ATT ATT C-3′ as a reverse primer instead, digested with SacI and XbaI, and cloned into pGEM4. Full-length Mgm1 was cloned into pYES2 (Promega) after amplification with the reverse primer 5′-CCC CGC GGC CGC TCT AGA TCA TAA ATT TTT GGA GAC GCC C-3′ and digestion with SacI and XbaI. For the construction of s-Mgm1* (Fig. 4A), the sequence for the first 167 amino acids of cytochrome b 2 was amplified using primers 5′-CCC CGA GCT CGC CAT GCT AAA ATA CAA ACC TTT ACT A-3′ and 5′-GAT GTA GCG GCT ATT AGA GTA GCG GAT CCT TGA AGG GGA CCC-3′ and fused to Mgm1-(161–902) by two-sided overlap extension PCR (20Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar) with the second PCR product obtained by amplification with 5′-GGG TCC CCT TCA AGG ATC CGC TAC TCT AAT AGC CGC TAC ATC-3′ and the reverse primer for full-length Mgm1. The resulting sequence coding for the fusion protein was cloned into pYES2 using the SacI and XbaI restriction sites. The large isoform, l-Mgm1* (Fig. 4A), was obtained in a similar way using 5′-CAC AAG ACG GTG GTC ATG GAT CAC TCG ACG ATG ACG AAA GT-3′ and 5′-ACT TTC GTC ATC GTC GAG TGA TCC ATG ACC ACC GTC TTG TG-3′ as the two overlapping internal primers carrying the deletion of residues 154–167 of Mgm1. For the expression of full-length Mgm1 and l-Mgm1* under control of the endogenous promoter, these constructs were subcloned into pRS315 (21Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 1-270PubMed Google Scholar) using the SacI and XbaI restriction sites. Approximately 1000 bp upstream and the first 351 bp of the Mgm1 open reading frame were amplified from genomic DNA using the primers 5′-CCC CGA GCT CCA AGT CAT GTG AAG GAT GGA C-3′ and 5′-CCC CGC TAG CTT CTT CCA TCT TAT AAG C-3′. The resulting fragment was exchanged for a fragment of the respective constructs in pRS315 using the SacI and NheI restriction sites. For s-Mgm1*, the promoter was amplified using 5′-CCC CGA ATT CAG ATC TTT AGT AAA GGT TTG TAT TTT AGC ATG CTT TCA GAG TAT TAT GGT GAA-3′ as the reverse primer instead, which introduces a BglII site into the sequence of cytochrome b 2 by a silent mutation and cloned into the SacI and EcoRI restriction sites of pYES2. s-Mgm1* was amplified from s-Mgm1* in the pYES2 plasmid using the primer 5′-CCC CAG ATC TCG AAG AAC TGT GAG GCT GC-3′, which also contains the silent mutation for the BglII restriction site and the reverse primer for full-length Mgm1. The resulting fragment was cloned into the BglII and XbaI restriction sites of pYES2 containing the endogenous promoter of Mgm1 and then subcloned into pRS313 (21Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 1-270PubMed Google Scholar) using the SacI and XbaI restriction sites. All constructs were verified by DNA sequencing. S. cerevisiae W303 (ade2–1, leu2–3, his3–11,15, trp1–1, ura3–1, can1–100 MATα) was used as a wild type strain. The plasmids with the endogenous promoter were used for transformation of the heterozygous diploid deletion strains MGM1/Δmgm1 or PCP1/Δpcp1 (obtained from EUROSCARF, Germany). Protease deletion strains (Fig. 3) were obtained from the homozygous diploid deletion library (Research Genetics). Mitochondria for in vitro import experiments were prepared from S. cerevisiae D273-10B. Culturing of yeast, sporulation of diploid strains, and tetrad dissection were performed according to standard protocols (22Sherman F.F. Fink G.R. Hicks J. Methods in Yeast Genetics: A Laboratory Course. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar).Fig. 3Pcp1 is required for the processing of l-Mgm1 to s-Mgm1. Total cell extracts of deletion mutants deficient in one known or predicted mitochondrial protease were prepared and analyzed by immunoblotting for Mgm1. Lack of s-Mgm1 in the Δpcp1 strain is indicated by an arrowhead. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Antibodies—Antibodies against Mgm1 were raised in rabbits using as antigens the C-terminal peptide H2N-CKKSYKGVSKNL-COOH and the internal peptide H2N-CSHQFEKAYFKENKK-COOH, both containing an additional cysteine for coupling to an affinity resin. Peptide synthesis, coupling of the peptide to keyhole limpet hemocyanin, and immunization of the rabbits were carried out by Pineda Antikörperservice (Berlin, Germany). For affinity purification, peptides were coupled to SulfoLink® Coupling Gel (Pierce) according to the manufacturer's instructions. The antibody against the C-terminal epitope was used for Western analysis unless indicated differently. Determination of Growth on Nonfermentable Carbon Source—In order to compare growth on fermentable versus nonfermentable carbon sources, drop dilution assays were performed. After tetrad dissection, cells were grown to exponential phase for 16 h on liquid-selective glucose medium at 30 °C, adjusted to a concentration of 0.7 A 578 nm/ml, and subjected to consecutive 10-fold dilution steps. 5-μl aliquots of each dilution were spotted on YPD and YPG plates in duplicate, and the plates were incubated for two (YPD) or three (YPG) days at 30 °C. Import of Preproteins into Mitochondria—Radiolabeled precursor proteins were synthesized using a coupled reticulocyte lysate transcription-translation system (Promega) in the presence of [35S]methionine. Mitochondria were isolated as described (23Sirrenberg C. Bauer M.F. Guiard B. Neupert W. Brunner M. Nature. 1996; 384: 582-585Crossref PubMed Scopus (255) Google Scholar). Import reactions were carried out in import buffer at 25 °C (600 mm sorbitol, 50 mm HEPES, 80 mm KCl, 10 mm MgAc2, 2.5 mm EDTA, 2 mm KH2PO4, 5 mm NADH, 2.5 mm ATP, 2.5 mm malate, 2.5 mm succinate, 0.1% bovine serum albumin, pH 7.2). 50 μg of mitochondria and 1% (v/v) reticulocyte lysate with the radiolabeled precursor were used per import reaction. Membrane potential was dissipated by adding carbonyl cyanide 3-chlorophenylhydrazone to a final concentration of 50 μm. After import, samples were diluted, treated with hypoosmotic buffer (20 mm HEPES/KOH, pH 7.4) to selectively rupture the outer membrane, and treated with proteinase K as indicated. Carbonate Extraction—To extract peripherally bound membrane proteins, mitochondria were diluted to a final concentration of 1 mg/ml in 20 mm HEPES/KOH, pH 7.4. After the addition of an equal volume of freshly prepared 0.2 m sodium carbonate solution, samples were incubated for 30 min at 4 °C. The membrane and soluble fractions were separated by centrifugation at 45,000 rpm in a TLA45 rotor for 30 min at 4 °C. Equal fractions of membrane-associated and soluble proteins were analyzed by SDS-PAGE and immunoblotting. Yeast Total Cell Extracts—Yeast total cell extracts were prepared by alkaline lysis. The pellet of 2 ml of yeast culture (A 578 = 1) was resuspended in 250 μl of 50 mm Tris/HCl, pH 8. Then 50 μl of lysis buffer (1.85 m NaOH, 7.4% (v/v) β-mercaptoethanol and 20 mm phenylmethylsulfonyl fluoride) were added. After incubation for 10 min at 4 °C, samples were precipitated with 220 μl of 72% (w/v) trichloroacetic acid, washed once with acetone, and analyzed by SDS-PAGE and Western blotting. Fluorescence Microscopy—Heterozygous diploid strains were cotransformed with plasmid pVT100U-mtGFP expressing mitochondria targeted green fluorescent protein (24Westermann B. Neupert W. Yeast. 2000; 16: 1421-1427Crossref PubMed Scopus (313) Google Scholar). After tetrad dissection, cells were grown for 16 h to exponential phase in liquid-selective glucose medium at 30 °C and analyzed by standard fluorescence microscopy (25Prokisch H. Neupert W. Westermann B. Mol. Biol. Cell. 2000; 11: 2961-2971Crossref PubMed Scopus (38) Google Scholar). Classification and quantification of the morphology phenotypes were performed without knowledge of strain identity at the time of analysis. To test for the presence of mitochondrial DNA, cells grown under the same conditions were stained with 1 μg/ml DAPI for 1 h and washed once with phosphate-buffered saline. Only cells showing no trace of staining outside the nucleus were classified as lacking mtDNA (rhoO). For quantification of the phenotype, at least 150 cells were analyzed in three independent experiments, and the average and S.D. were calculated. Determination of N Termini of Mgm1 Isoforms—10 mg of mitochondria were lysed in 10 mm Tris/HCl, pH 7.6, 0.5% (w/v) Triton X-100, 150 mm NaCl, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride for 15 min. After a clarifying spin, the supernatant was subjected to immunoprecipitation for 3 h at 4 °C using Protein A-Sepharose beads (Amersham Biosciences) preloaded with antibodies against the C terminus of Mgm1. Samples were eluted from the beads with SDS-containing buffer, separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride membrane. Mgm1 bands were cut and subjected to N-terminal sequencing by Edman degradation (TOPLAB GmbH, Germany). Localization of Mgm1—In a first approach to determine the submitochondrial location of Mgm1, two different derivatives of precursors of Mgm1, Mgm1-(1–228)-dihydrofolate reductase and Mgm1-(1–427), were imported into isolated yeast mitochondria (Fig. 1). Both precursors were processed upon import, yielding proteins of 47 kDa (Fig. 1A) and 43 kDa (Fig. 1B), respectively. This corresponds to the removal of the N-terminal 9-kDa mitochondrial targeting sequence of Mgm1 by the mitochondrial processing peptidase (MPP). When proteinase K was added after import, the mature proteins were protected from digestion in mitochondria, indicating that the precursors were transported across the outer membrane. When the outer membrane was selectively opened by hypoosmotic shock (swelling), the imported and processed proteins became sensitive to proteinase K. The inner membrane was not ruptured after swelling, since matrix proteins were not degraded and the ADP/ATP carrier (Aac2) was converted to a fragment indicative of proteolytic attack from the intermembrane space (data not shown). Taken together, both Mgm1 precursor proteins became localized in the intermembrane space after import in vitro. As expected for proteins containing an N-terminal mitochondrial targeting sequence, the import of both precursors was dependent on the presence of membrane potential, ΔΨ. We conclude that residues 1–228 are sufficient to localize Mgm1 to the intermembrane space. In a second approach, we studied the submitochondrial distribution of endogenous Mgm1. Isolated yeast mitochondria were treated with proteinase K and subjected to SDS-electrophoresis and immunoblotting using antibodies directed against a C-terminal (Fig. 1C) and an internal epitope of Mgm1 (Fig. 1D). Endogenous Mgm1 was protected from digestion by proteinase K in mitochondria in both cases. After selective opening of the outer membrane, both isoforms of Mgm1 were digested by the protease. Swelling efficiency and the intactness of the inner membrane after swelling were controlled by decorating the same Western blots with antibodies against Oxa1 and Aac2; fragments characteristic for mitochondria with opened outer membrane and intact inner membrane were observed (26Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (133) Google Scholar, 27Pfanner N. Neupert W. J. Biol. Chem. 1987; 262: 7528-7536Abstract Full Text PDF PubMed Google Scholar). Taken together, the C terminus and the internal epitope (residues 484–497) of both Mgm1 isoforms are located in the intermembrane space. This is consistent with the localization proposed by Wong et al. (2Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. Nunnari J. J. Cell Biol. 2000; 151: 341-352Crossref PubMed Scopus (269) Google Scholar) and by two recent reports (3Wong E.D. Wagner J.A. Scott S.V. Okreglak V. Holewinske T.J. Cassidy-Stone A. Nunnari J. J. Cell Biol. 2003; 160: 303-311Crossref PubMed Scopus (196) Google Scholar, 4Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in pressGoogle Scholar). Also, the human ortholog OPA1 was recently localized to this subcompartment (17Olichon A. Emorine L.J. Descoins E. Pelloquin L. Brichese L. Gas N. Guillou E. Delettre C. Valette A. Hamel C.P. Ducommun B. Lenaers G. Belenguer P. FEBS Lett. 2002; 523: 171-176Crossref PubMed Scopus (335) Google Scholar). In summary, both isoforms of Mgm1, the large isoform (l-Mgm1; 97 kDa) and the short isoform (s-Mgm1; 84 kDa) are located in the intermembrane space. Determination of the N Termini of Both Mgm1 Isoforms—In order to further analyze the structure of the two isoforms, their N termini were determined. To this end, mitochondria were solubilized, and Mgm1 was immunoprecipitated using an antibody raised against the C terminus. After SDS-PAGE and blotting onto a polyvinylidene difluoride membrane, the bands corresponding to the two isoforms were cut out from the membrane, and the respective N termini were determined by Edman degradation (Fig. 2A). In the case of l-Mgm1, the N-terminal sequence was ISHFPKII, corresponding to amino acid residues 81–88 of Mgm1. Since there is an MPP consensus site (28Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (981) Google Scholar) after residue Ser80, we conclude that l-Mgm1 is generated by MPP cleavage immediately after the mitochondrial targeting sequence of Mgm1. In the case of s-Mgm1, the two peptides ATLIAATS and LIAATS were found in similar amounts. These peptides correspond to amino acid residues 161–168 and 163–168, respectively. We suggest that the initial cleavage takes place after Thr160 and that the further removal of two residues is caused by other peptidases in the intermembrane space or during the preparation of cell extracts. Still, the possibility is not excluded that the initial cleavage of Mgm1 can occur after Thr160 as well as after Thr162. Membrane Association of Mgm1—Mgm1 contains a predicted transmembrane segment from residue 94 to 113, which, as shown above, is only present in l-Mgm1 and not in s-Mgm1. Therefore, the two isoforms should differ in their membrane association. In order to address this, mitochondria were swollen to rupture the outer membrane and then extracted with either low salt, high salt, or sodium carbonate, and membranes were separated by ultracentrifugation. Neither of the two isoforms of Mgm1 was released from mitochondria by swelling alone, indicating that they are not soluble proteins in the intermembrane space (Fig. 2B). With high salt, about 50% of s-Mgm1 was released from the membranes, whereas l-Mgm1 was resistant to salt extraction (Fig. 2B). Treatment with sodium carbonate led to the removal of significant fractions of both isoforms from the membrane (Fig. 2B). However, l-Mgm1 was more resistant, probably because it contains the predicted N-terminal membrane-spanning segment. The incomplete resistance of l-Mgm1 to carbonate extraction can be attributed to the presence of only few amino acid residues on the N-terminal side of the predicted transmembrane segment, leading to a weaker embedding in the membrane. A similar behavior upon alkaline treatment of mitochondria was observed for d-lactate dehydrogenase (Fig. 2B), a protein that is anchored to the inner membrane by a single N-terminal transmembrane segment and faces the intermembrane space (29Rojo E.E. Guiard B. Neupert W. Stuart R.A. J. Biol. Chem. 1998; 273: 8040-8047Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In contrast, the ADP/ATP carrier (Aac2), an integral membrane protein with six membrane-spanning segments (27Pfanner N. Neupert W. J. Biol. Chem. 1987; 262: 7528-7536Abstract Full Text PDF PubMed Google Scholar), was completely resistant to extraction by carbonate (Fig. 2B). In order to investigate whether l-Mgm1 and s-Mgm1 differ in their association with either the outer or the inner membrane, we generated mitochondrial membrane vesicles by sonication and fractionated them on a sucrose gradient. Both isoforms of Mgm1 were found to be associated with vesicles derived from both membranes (data not shown). This may be due to the release of Mgm1 from membranes during sonication and subsequent nonspecific binding to vesicles. Such an association with membranes would not be unusual for a dynamin-like protein, since dynamins are known to bind to and spontaneously assemble on lipid vesicles (30Sweitzer S.M. Hinshaw J.E. Cell. 1998; 93: 1021-1029Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). Unfortunately, this behavior makes it highly unreliable to conclude to which membrane Mgm1 is attached. The Mitochondrial Protease Pcp1 Is Involved in the Generation of s-Mgm1—In order to investigate how the isoforms of Mgm1 are generated, we prepared total cell extracts of different deletion mutants that were deficient in one of the known or putative mitochondrial proteases. In all strains, similar steady state levels of both isoforms of Mgm1 as in wild type were observed, with the exception of the Δpcp1 strain (Fig. 3). In this strain, the band corresponding to s-Mgm1 was absent, whereas the steady state level of l-Mgm1 was increased. Two additional minor bands were observed in the Δpcp1 strain, but they were distinct from s-Mgm1 in size. They are probably the result of nonspecific degradation during the preparation of cell extracts. Thus, the presence of Pcp1 appears to be essential for the generation of s-Mgm1. Pcp1 was shown to be imported into mitochondria in vitro (31Steinmetz L.M. Scharfe C. Deutschbauer A.M. Mokranjac D. Herman Z.S. Jones T. Chu A.M. Giaever G. Prokisch H. Oefner P.J. Davis R.W. Nat. Genet. 2002; 31: 400-404Crossref PubMed Scopus (433) Google Scholar). Furthermore, Pcp1 was identified in a recent screen for mutants with aberrant mitochondrial morphology and predicted to be located in the inner membrane of mitochondria (32Dimmer K.S. Fritz S. Fuchs F. Messerschmitt M. Weinbach N. Neupert W. Westermann B. Mol. Biol. Cell. 2002; 13: 847-853Crossref PubMed Scopus (352) Google Scholar). In that study, the Δpcp1 strain showed a phenotype strikingly similar to that of the Δmgm1 strain; both strains have fragmented mitochondria and cannot grow on nonfermentable carbon sources (10Guan K. Farh L. Marshall T.K. Deschenes R.J. Curr. Genet. 1993; 24: 141-148Crossref PubMed Scopus (116) Google Scholar, 11Jones B.A. Fangman W.L. Genes Dev. 1992; 6: 380-389Crossref PubMed Scopus (193) Google Scholar). The Δmgm1 strain is rho0, since it has lost mtDNA (11Jones B.A. Fangman W.L." @default.
• W2079676584 created "2016-06-24" @default.
• W2079676584 creator A5002414711 @default.
• W2079676584 creator A5006765832 @default.
• W2079676584 creator A5040462964 @default.
• W2079676584 creator A5046289240 @default.
• W2079676584 creator A5053562075 @default.
• W2079676584 date "2003-07-01" @default.
• W2079676584 modified "2023-10-01" @default.
• W2079676584 title "Processing of Mgm1 by the Rhomboid-type Protease Pcp1 Is Required for Maintenance of Mitochondrial Morphology and of Mitochondrial DNA" @default.
• W2079676584 cites W1489237559 @default.
• W2079676584 cites W1493752155 @default.
• W2079676584 cites W1507851645 @default.
• W2079676584 cites W1689925871 @default.
• W2079676584 cites W1862613154 @default.
• W2079676584 cites W1924863471 @default.
• W2079676584 cites W1972812883 @default.
• W2079676584 cites W1979328799 @default.
• W2079676584 cites W1985634923 @default.
• W2079676584 cites W1986996580 @default.
• W2079676584 cites W1998591058 @default.
• W2079676584 cites W1999053124 @default.
• W2079676584 cites W2003346050 @default.
• W2079676584 cites W2012563825 @default.
• W2079676584 cites W2032220731 @default.
• W2079676584 cites W2033062319 @default.
• W2079676584 cites W2033462967 @default.
• W2079676584 cites W2039340205 @default.
• W2079676584 cites W2041001616 @default.
• W2079676584 cites W2051338661 @default.
• W2079676584 cites W2052074631 @default.
• W2079676584 cites W2064528123 @default.
• W2079676584 cites W2072087702 @default.
• W2079676584 cites W2077244062 @default.
• W2079676584 cites W2090866135 @default.
• W2079676584 cites W2091214639 @default.
• W2079676584 cites W2092705523 @default.
• W2079676584 cites W2113946252 @default.
• W2079676584 cites W2120965886 @default.
• W2079676584 cites W2128916568 @default.
• W2079676584 cites W2135588013 @default.
• W2079676584 cites W2135853075 @default.
• W2079676584 cites W2139963282 @default.
• W2079676584 cites W2141771296 @default.
• W2079676584 cites W2157920366 @default.
• W2079676584 cites W2462855682 @default.
• W2079676584 cites W4376848558 @default.
• W2079676584 doi "https://doi.org/10.1074/jbc.m211311200" @default.
• W2079676584 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12707284" @default.
• W2079676584 hasPublicationYear "2003" @default.
• W2079676584 type Work @default.
• W2079676584 sameAs 2079676584 @default.
• W2079676584 citedByCount "351" @default.
• W2079676584 countsByYear W20796765842012 @default.
• W2079676584 countsByYear W20796765842013 @default.
• W2079676584 countsByYear W20796765842014 @default.
• W2079676584 countsByYear W20796765842015 @default.
• W2079676584 countsByYear W20796765842016 @default.
• W2079676584 countsByYear W20796765842017 @default.
• W2079676584 countsByYear W20796765842018 @default.
• W2079676584 countsByYear W20796765842019 @default.
• W2079676584 countsByYear W20796765842020 @default.
• W2079676584 countsByYear W20796765842021 @default.
• W2079676584 countsByYear W20796765842022 @default.
• W2079676584 countsByYear W20796765842023 @default.
• W2079676584 crossrefType "journal-article" @default.
• W2079676584 hasAuthorship W2079676584A5002414711 @default.
• W2079676584 hasAuthorship W2079676584A5006765832 @default.
• W2079676584 hasAuthorship W2079676584A5040462964 @default.
• W2079676584 hasAuthorship W2079676584A5046289240 @default.
• W2079676584 hasAuthorship W2079676584A5053562075 @default.
• W2079676584 hasBestOaLocation W20796765841 @default.
• W2079676584 hasConcept C104317684 @default.
• W2079676584 hasConcept C153911025 @default.
• W2079676584 hasConcept C181199279 @default.
• W2079676584 hasConcept C182220744 @default.
• W2079676584 hasConcept C185592680 @default.
• W2079676584 hasConcept C24586158 @default.
• W2079676584 hasConcept C28859421 @default.
• W2079676584 hasConcept C499950583 @default.
• W2079676584 hasConcept C50729890 @default.
• W2079676584 hasConcept C54355233 @default.
• W2079676584 hasConcept C552990157 @default.
• W2079676584 hasConcept C55493867 @default.
• W2079676584 hasConcept C86803240 @default.
• W2079676584 hasConcept C95444343 @default.
• W2079676584 hasConceptScore W2079676584C104317684 @default.
• W2079676584 hasConceptScore W2079676584C153911025 @default.
• W2079676584 hasConceptScore W2079676584C181199279 @default.
• W2079676584 hasConceptScore W2079676584C182220744 @default.
• W2079676584 hasConceptScore W2079676584C185592680 @default.
• W2079676584 hasConceptScore W2079676584C24586158 @default.
• W2079676584 hasConceptScore W2079676584C28859421 @default.
• W2079676584 hasConceptScore W2079676584C499950583 @default.
• W2079676584 hasConceptScore W2079676584C50729890 @default.
• W2079676584 hasConceptScore W2079676584C54355233 @default.
• W2079676584 hasConceptScore W2079676584C552990157 @default.
• W2079676584 hasConceptScore W2079676584C55493867 @default.

• next