Crucial Mitochondrial Impairment upon CDC48 Mutation in Apoptotic Yeast
2006; Elsevier BV; Volume: 281; Issue: 35 Linguagem: Inglês
10.1074/jbc.m513699200
ISSN1083-351X
AutoresRalf J. Braun, Hans Zischka, Frank Madeo, Tobias Eisenberg, Silke Wissing, Sabrina Büttner, Silvia Engelhardt, Dietmute Büringer, Marius Ueffing,
Tópico(s)Mitochondrial Function and Pathology
ResumoMutation in CDC48 (cdc48S565G), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48S565G yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48S565G cells. Mitochondrial dysfunction in the cdc48S565G strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis. Mutation in CDC48 (cdc48S565G), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48S565G yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48S565G cells. Mitochondrial dysfunction in the cdc48S565G strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis. Fundamental cellular processes, such as the formation of organelles (ER, 3The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; VCP, valosin-containing protein; EM, electron microscopy; 2-DE, two-dimensional gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ROS, reactive oxygen species; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; FMK, fluoromethyl ketone; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MMF1, maintenance of mitochondrial function 1; MRP8, mitochondrial 40 S ribosomal protein; NE, nuclear envelope; T, total percentage concentration of acrylamide and bisacrylamide monomers.3The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; IBMPFD, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia; VCP, valosin-containing protein; EM, electron microscopy; 2-DE, two-dimensional gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ROS, reactive oxygen species; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; FMK, fluoromethyl ketone; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MMF1, maintenance of mitochondrial function 1; MRP8, mitochondrial 40 S ribosomal protein; NE, nuclear envelope; T, total percentage concentration of acrylamide and bisacrylamide monomers. Golgi apparatus, and the nuclear envelope), or ubiquitin-dependent ER-associated protein degradation (ERAD) have been linked to the yeast protein Cdc48p and its highly conserved mammalian orthologue VCP (1Elkabetz Y. Shapira I. Rabinovich E. Bar-Nun S. J. Biol. Chem. 2004; 279: 3980-3989Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 2Ye Y. Meyer H.H. Rapoport T.A. Nature. 2001; 414: 652-656Crossref PubMed Scopus (889) Google Scholar, 3Richly H. Rape M. Braun S. Rumpf S. Hoege C. Jentsch S. Cell. 2005; 120: 73-84Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 4Woodman P.G. J. Cell Sci. 2003; 116: 4283-4290Crossref PubMed Scopus (140) Google Scholar). Mutations in VCP have been associated with "inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia" (IBMPFD), a dominant human disorder (5Watts G.D. Wymer J. Kovach M.J. Mehta S.G. Mumm S. Darvish D. Pestronk A. Whyte M.P. Kimonis V.E. Nat. Genet. 2004; 36: 377-381Crossref PubMed Scopus (1099) Google Scholar, 6Schroder R. Watts G.D. Mehta S.G. Evert B.O. Broich P. Fliessbach K. Pauls K. Hans V.H. Kimonis V. Thal D.R. Ann. Neurol. 2005; 57: 457-461Crossref PubMed Scopus (152) Google Scholar). A genetic screening of a Drosophila model for human polyglutamine diseases, a class of inherited neurodegenerative disorders, identified the Drosophila homologue of Cdc48p/VCP as a modulator of apoptotic cell death (7Higashiyama H. Hirose F. Yamaguchi M. Inoue Y.H. Fujikake N. Matsukage A. Kakizuka A. Cell Death Differ. 2002; 9: 264-273Crossref PubMed Scopus (111) Google Scholar), leading these authors to propose VCP as a pathological effector for polyglutamine-induced neurodegeneration. However, the cellular mechanisms underlying VCP-mediated cell death in these human disorders remain largely unknown.Apoptotic phenotypes in cells expressing mutated Cdc48p/VCP have originally been described in budding yeast (8Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (677) Google Scholar) and were thereafter confirmed in mammalian cell cultures (9Shirogane T. Fukada T. Muller J.M. Shima D.T. Hibi M. Hirano T. Immunity. 1999; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 10Hirabayashi M. Inoue K. Tanaka K. Nakadate K. Ohsawa Y. Kamei Y. Popiel A.H. Sinohara A. Iwamatsu A. Kimura Y. Uchiyama Y. Hori S. Kakizuka A. Cell Death Differ. 2001; 8: 977-984Crossref PubMed Scopus (234) Google Scholar), in trypanosomes (11Lamb J.R. Fu V. Wirtz E. Bangs J.D. J. Biol. Chem. 2001; 276: 21512-21520Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and in zebrafish (12Imamura S. Ojima N. Yamashita M. FEBS Lett. 2003; 549: 14-20Crossref PubMed Scopus (34) Google Scholar). Notably, Cdc48p was the first apoptotic mediator found in Saccharomyces cerevisiae (8Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (677) Google Scholar). The expression of a point-mutated CDC48 gene (cdc48S565G) leads to a characteristic apoptotic phenotype: phosphatidylserine externalization, DNA fragmentation, chromatin condensation, nuclear fragmentation, and vacuolization (8Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (677) Google Scholar, 13Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (860) Google Scholar). These results obtained in the cdc48S565G strain initiated the establishment of yeast as a model to study evolutionary conserved mechanisms of apoptotic regulation (14Ludovico P. Madeo F. Silva M. IUBMB Life. 2005; 57: 129-135Crossref PubMed Scopus (60) Google Scholar, 15Madeo F. Herker E. Wissing S. Jungwirth H. Eisenberg T. Frohlich K.U. Curr. Opin. Microbiol. 2004; 7: 655-660Crossref PubMed Scopus (256) Google Scholar, 16Weinberger M. Ramachandran L. Burhans W.C. IUBMB Life. 2003; 55: 467-472Crossref PubMed Scopus (21) Google Scholar).Mitochondria play a crucial role in many apoptotic pathways in both mammalian cells and in yeast (17Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2775) Google Scholar, 18Ludovico P. Rodrigues F. Almeida A. Silva M.T. Barrientos A. Corte-Real M. Mol. Biol. Cell. 2002; 13: 2598-2606Crossref PubMed Scopus (318) Google Scholar, 19Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1069) Google Scholar). In the present study, we therefore tested for mitochondrial impairment and contribution in Cdc48p-mediated apoptosis. We observed mitochondrial enlargement, distinct alterations in the mitochondrial proteome, release of cytochrome c into the cytosol, impairment in the ability of cdc48S565G cells to adapt to respiratory conditions, as well as mitochondrial ROS production paralleled to the emergence of caspase-like enzymatic activity. These data show mitochondrial impairment at morphological, molecular, and functional levels. These alterations are associated with apoptotic cell death indicating the activation of a mitochondrial pathway for Cdc48p-mediated apoptosis.EXPERIMENTAL PROCEDURESYeast Strains, Culture Conditions, and Assay for Respiratory Deficiency—S. cerevisiae wild-type KFY417 (CDC48) and mutant strain KFY437 (cdc48S565G) (20Madeo F. Schlauer J. Frohlich K.U. Gene (Amst.). 1997; 204: 145-151Crossref PubMed Scopus (18) Google Scholar) were used in this study. For all experiments (except ρ0/ρ+ experiments, see below) induction of apoptosis was done as follows (8Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (677) Google Scholar, 13Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (860) Google Scholar): glucose medium (YP medium, 1% yeast extract, 2% Bacto Peptone, supplemented with 4% glucose, Otto Nordwald, Hamburg, Germany) was inoculated (A600 = 0.1-0.3) with stationary YPGal pre-cultures (YP medium supplemented with 4% galactose). Cells were then grown in baffled flasks at 28 °C until early stationary and stationary phases, respectively, and then subjected to heat shock at 37 °C.For analysis of respiratory deficiency, glucose cultures of both wild-type and cdc48S565G strains were plated on YP plates (YP medium supplemented with 1.5% agar) containing (i) 4% glucose (YPGlc, fermentative selective medium) or (ii) 2% lactate (YPLac, respiratory selective medium). Cultures were spotted on agar plates in dilution series (from 5 × 106 cells to 5 × 101 cells in 10-fold dilution steps) clockwise on six distinct sections. After 5 days of incubation at room temperature, the sections were evaluated for growth.ρ0 strains (yeast strains lacking functional mitochondria) were generated from the respective ρ+ strains (KFY417 and KFY437) by growing cells in media containing 10 μg/ml ethidium bromide for 3 days. The resulting respiratory deficiency was confirmed by complete lack of growth on obligatory respiratory media (glycerol). In ρ0/ρ+ experiments, cells were grown and treated as described for KFY417 and KFY437 (see above) with the modification that pre-cultures were grown in YPGal/Glc (3% galactose/1% glucose), because the generated ρ0 strains were unable to grow in YPGal.Electron Microscopy—EM analysis of mitochondrial samples was carried out as previously described (21Zischka H. Weber G. Weber P.J. Posch A. Braun R.J. Buhringer D. Schneider U. Nissum M. Meitinger T. Ueffing M. Eckerskorn C. Proteomics. 2003; 3: 906-916Crossref PubMed Scopus (126) Google Scholar). EM analysis of stationary yeast cells to visualize membrane structures was done essentially according to Ref. 22Byers B. Goetsch L. Guthrie C. Guide to Yeast Genetics and Molecular Biology. Academic Press, San Diego, CA1991: 603-626Google Scholar: cells were harvested and incubated for 8 min in fixative (4% formaldehyde, 2% glutaraldehyde, 4% sucrose, 2 mm calcium acetate, 50 mm sodium cacodylate, pH 7.2) at room temperature. Fixed cells were stored in fixative overnight at 4 °C and subsequently prepared for cell wall removal by incubation in pretreatment solution (0.2 m Tris/HCl, 100 mm β-mercaptoethanol) for 10 min at room temperature. Removal of cell wall was done with 30 units of lyticase (Sigma) and 0.6 unit of arylsulfatase (Roche Applied Science) for 90 min at 30 °C in digestion buffer (35 mm potassium phosphate buffer, pH 6.8, 0.5 mm MgCl2, 1.2 m sorbitol). Cells were washed in cacodylate buffer (0.1 m sodium cacodylate, 5 mm CaCl2), postfixed (0.5% osmium tetroxide, 0.8% potassium ferrocyanide), washed in distilled water, stained en bloc (1% aqueous uranyl acetate), dehydrated in ascending alcohol series, and embedded in Araldite. The preparations were sectioned at 50 nm on an ultramicrotome (Ultrotom III, LKB, Bromma, Sweden), and EM micrographs were obtained on a Zeiss (Oberkochen, Germany) EM 10 electron microscope.Cell Fractionation—Mitochondria were isolated by differential centrifugation as described in Zischka et al. (21Zischka H. Weber G. Weber P.J. Posch A. Braun R.J. Buhringer D. Schneider U. Nissum M. Meitinger T. Ueffing M. Eckerskorn C. Proteomics. 2003; 3: 906-916Crossref PubMed Scopus (126) Google Scholar). Cytosol was obtained by ultracentrifugation (177,000 × g, 90 min, 4 °C) from the supernatant of the first mitochondrial sedimentation.Two-dimensional Gel Electrophoresis and Image Analysis— 2-DE was performed according to Zischka et al. (21Zischka H. Weber G. Weber P.J. Posch A. Braun R.J. Buhringer D. Schneider U. Nissum M. Meitinger T. Ueffing M. Eckerskorn C. Proteomics. 2003; 3: 906-916Crossref PubMed Scopus (126) Google Scholar). Isoelectric focusing was done using immobilized pH-gradient strips (pH 3-10 non-linear) and gradient gels (8-16% T) for SDS-PAGE. Resultant protein patterns were detected by standard staining procedures, either silver (23Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar) for analytical purposes (150 μg of protein per gel) or "ruthenium II Tris bathophenanthroline disulfonate fluorescent dye" (24Rabilloud T. Strub J.M. Luche S. van Dorsselaer A. Lunardi J. Proteomics. 2001; 1: 699-704Crossref PubMed Scopus (287) Google Scholar) for preparative purposes (400 μg of protein per gel). Gels treated with the latter were further stained with colloidal Coomassie Blue for protein analysis (25Neuhoff V. Arold N. Taube D. Ehrhardt W. Electrophoresis. 1988; 9: 255-262Crossref PubMed Scopus (2342) Google Scholar). Image analysis of the gels was done with the ProteomWeaver™ image analysis software V.2.2 (Definiens AG, Munich, Germany). For the analysis of mitochondrial extracts data were determined by taking into account three independent experiments.Protein Identification via MALDI-TOF Mass Spectrometry— Proteins were subjected to a sequence-dependent protease treatment (100 ng of trypsin per gel plug, Promega, Mannheim, Germany) as described by Shevchenko et al. (23Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar). Resulting peptides were analyzed by peptide mass fingerprinting with the thin layer method (26Kussmann M. Roepstorff P. Methods Mol. Biol. 2000; 146: 405-424PubMed Google Scholar) using a MALDI-TOF Reflectron (Waters, Eschborn, Germany). Data base searches for protein identification were done in SwissProt using the ProteinLynx Globalserver 1.1 software (PLGS 1.1, Waters).SDS-PAGE and Immunoblotting Analysis—SDS-PAGE and subsequent immunoblotting on polyvinylidene difluoride membranes were carried out according to standard procedures. Immunoblots were incubated with anti-55 kDa cytosolic protein (kind gift of G. Blobel) and anti-cytochrome c (kind gift of F. Sherman), respectively. Immunoreactive bands were visualized by ECL plus (GE Healthcare, Freiburg, Germany) and quantified using QuantityOne® V.4.2 software (Bio-Rad, Munich, Germany).Staining for Reactive Oxygen Species—ROS were detected with dihydrorhodamine 123 (Sigma) according to Madeo et al. (13Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (860) Google Scholar) with 30-min staining at 30 °C. Cells were embedded in 0.5% agarose in PBS and evaluated for staining by fluorescence microscopy using a rhodamine optical filter (room temperature, 40×/0.75, Axioskop 2, AxioCam HRc, AxioVision 4, Zeiss, Göttingen, Germany). In ρ0/ρ+ experiments, ROS were detected with the mitochondrial membrane potential-independent stain dihydroethidium (Sigma). 5 × 106 cells were pelleted in 96-well microtiter plates (Microlon Fluorotrac 600, Greiner, Austria), washed twice with PBS, resuspended in 250 μl of 2.5 μg/ml dihydroethidium in PBS, and incubated for 10 min at room temperature. Relative fluorescence units were determined using a fluorescence reader (GENios Pro™, Tecan, Grödig, Austria, excitation 515 nm, emission 595 nm, room temperature). Dihydroethidium was used as the blank in PBS. Additionally, cells were evaluated for staining by fluorescence microscopy using a rhodamine optical filter.Survival Plating Assay—Survival plating assays were done as previously described (27Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). Briefly, an aliquot of the culture was counted with a CASY1 (Schärfe Systems, Reutlingen, Germany), diluted 1:10,000 in water, and 500 cells were plated on YPGlc plates (4% glucose). The number of colonies (colony forming units) was determined after incubating the plates for 2-3 days at 28 °C. For each experiment three plates per strain and condition were evaluated for growth of colonies.Tests for Apoptotic Markers—In vivo measurement of caspase-like enzymatic activity by flow cytometric analysis was done as previously described (27Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar). Briefly, cells were harvested, washed in PBS, and resuspended in staining solution containing fluorescein isothiocyanate (FITC)-VAD-FMK (CaspACE™, Promega). After incubation for 20 min at 30 °C, cells were washed and resuspended in PBS. Stained cells were counted using a FACSCalibur (BD Biosciences) and Cell Quest analysis software. CaspACE™ FITC-VAD-FMK in situ marker is an FITC conjugate of the cell-permeable caspase inhibitor VAD-FMK. This structure allows delivery of the inhibitor into the cell where it binds to activated caspase, serving as an in situ marker for apoptosis. The bound marker is localized by fluorescence detection.The T4 terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to visualize DNA fragmentation, a late marker of apoptosis. Cell wall digestion and cell fixation were done as described by Madeo et al. (8Madeo F. Frohlich E. Frohlich K.U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (677) Google Scholar). TUNEL reaction was performed using an in situ cell death detection kit (Roche Applied Science) and Chromatide Bodipy™ (Molecular Probes, Invitrogen, Karlsruhe, Germany) as fluorescence-labeled dUTP. Cells were evaluated for stained nuclei by fluorescence microscopy using a FITC optical filter (room temperature, 40×/0.75, Axioskop 2, AxioCam HRc, AxioVision 4).RESULTSMitochondria in cdc48S565G Cells Are Enlarged Compared with Wild-type—To check for mitochondrial impairment in the apoptotic cdc48S565G yeast strain we performed ultrastructural analysis (EM) of yeast cells. In cdc48S565G cells we observed a significant enlargement of mitochondria compared with wild type (Fig. 1A, for quantification see Fig. 1B). In the cdc48S565G strain 10% of the cellular area was composed of mitochondria compared with 7% in the wild-type strain. Because the average number of mitochondria per cell was highly similar between the cdc48S565G and wild-type strains (1.1 for wild-type and 1.2 for cdc48S565G cells), these data hint toward a swelling of mitochondria in the cdc48S565G strain, which is a known feature in pathophysiological processes (28Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (655) Google Scholar, 29Boya P. Cohen I. Zamzami N. Vieira H.L. Kroemer G. Cell Death Differ. 2002; 9: 465-467Crossref PubMed Scopus (117) Google Scholar, 30Farber J.L. Environ. Health Perspect. 1994; 102: 17-24Crossref PubMed Scopus (291) Google Scholar, 31Wakabayashi T. Acta Biochim. Pol. 1999; 46: 223-237Crossref PubMed Scopus (28) Google Scholar).Distinct Alterations Are Observed in the Mitochondrial Proteome of cdc48S565G Cells Compared with Wild-type—We further investigated whether mitochondrial enlargement in the cdc48S565G strain was concomitant with alterations at the molecular level of mitochondria. Therefore, we analyzed the mitochondrial proteome applying differential 2-DE analysis of wild-type and cdc48S565G strains. Additionally, we compared their total cell extracts and their cytosolic proteomes.Differential 2-DE analysis of mitochondria resulted in 32 significant protein spot variations between wild-type and cdc48S565G strains (Fig. 2A, compare gels 3 and 4, and Table 1). In contrast, only minimal differences were observed in cytosolic fractions (Fig. 2A, compare gels 1 and 2), and the overall cellular proteome remained unchanged (data not shown).FIGURE 2Differential 2-DE analysis of mitochondrial and cytosolic fractions from wild-type and cdc48S565G cells. A, 2-DE comparison of cytosolic extracts (wild-type versus cdc48S565G, gels 1 and 2, respectively): 6 reproducible differences (arrows) out of 1600 protein spots per gel were found (Proteom Weaver™) (n = 6). Comparison of mitochondrial extracts (gels 3 and 4): 32 reproducible differences (arrows) out of 1400 protein spots per gel were found; identified proteins and results of quantification (ProteomWeaver™) are listed in Table 1 (n = 7). B, representative differences between wild-type and cdc48S565G strains in mitochondrial extracts.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Identified proteins differentially found in mitochondrial extracts For MALDI-TOF mass spectrometry, protein spots were subjected to trypsin treatment. Resulting peptides were analyzed by peptide mass fingerprinting using a MALDI-TOF Reflection (Waters). Spectra were annotated applying MassLynx software (Waters). Subsequent data base searches in SwissProt were done using the ProteinLynx Globalserver 1.1 software (PLGS 1.1, Waters) with the following search parameters: organisms, unrestricted; fixed modifications, carbamidomethyl (C); variable modifications, oxidations (M); mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance, ±150 ppm; peptide charge state, 1+; maximum missed cleavages, 1. For 2-DE analysis of mitochondria, image analysis of the gels was performed by using ProteomWeaver™ image analysis software V.2.2 (Definiens). For the analysis of mitochondrial extracts data were determined by taking into account three independent experiments. In total seven two-dimensional gels for wild-type and seven two-dimensional gels for cdc48S565G were considered for quantification and statistics.Spot no. (Fig. 3)SwissProt Accession no.aSwissProt data base: us.expasy.org/sprot/Sequence coveragePLGS 1.1 score of identified protein/score of next yeast hitbPLGS 1.1 scoringMatched mass valuesGene name (MIPS)cMIPS database: mips.gsf.de/projects/fungiProtein nameRelative protein spot intensities on 2-DE (cdc48S565G versus wild type)dFactor: mutant (cdc48S565G) versus wild-type strain: 1 for accumulation of proteinp values of 2-DE image analysis (Student's t test)Localization (Mitop2)eMitop2 data base: ihg.gsf.de/mitop2/start.jspFunction%1Q012174254/269/13YER069wARG5, 6 protein0.50.00200Mitochondria, matrixAmino acid metabolism2P0616896201/5713/13YLR355cKetol acid reductoisomerase (Ilv5p)0.60.00332MitochondriaAmino acid metabolism3, 4P401858282/—7/9YIL051cMMF10.40.00361Mitochondria, matrixAmino acid metabolism5P257196549/148/11YML078wCyclophilin C0.70.00247Mitochondria, matrixProtein folding6P357194832/343/6YKL142wMRP84.00.01214MitochondriaProtein biosynthesis7, 8P080677874/479/17YEL024wUbiquinol-cytochrome c reductase iron-sulfur subunit (UCRI)0.65/1.20.04284/0.17448Mitochondria, inner mitochondrial membraneEnergy metabolism9P532526921/34/6YGR086cHypothetical protein YGR086c/sphingolipid long-chain base-responsive protein PIL13.50.00007MitochondriafAccording to Ref. 54, high probability for mitochondrial localization according to Mitop2 data base, lipid particlesMembrane traffic10P332047828/103/6YKL013cARP2/3 complex 20-kDa subunit3.50.00038CytoskeletonMitochondrial motility11P284953531/127/10YKL007wF-actin capping protein α subunit (CAPA)2.80.00020CytoskeletonActin cytoskeleton12P356914913/123/7YKL056cTranslationally controlled tumor protein homolog (TCTP)/microtubule and mitochondria interacting protein (Mmi1p)gAccording to Ref. 553.30.00037Cytoskeleton, mitochondria-associatedgAccording to Ref. 55Microtubule-associated, translocates to mitochondria upon oxidative stressgAccording to Ref. 5513P397425511/72/5YLR292cTranslocation protein Sec72p1.80.00460ER membraneSecretory pathway14, 15P072834946/—6/19YFL045cPhosphomannomutase Sec53p1.80.00014ER-associatedhAccording to Ref. 56Secretory pathway16P351767522/—17/13YDR304cCyclophilin D0.50.00291ER lumenProtein folding17—20P159925328/206/8YBR072wHeat shock protein 26 (Hsp26)1.70.00106Nucleus, cytoplasmStress response21P38011690/-64/10YMR116cGuanine nucleotide-binding protein subunit beta-like protein2.10.00154RibosomeiRibosomal proteins are predominantly NE-ER-associated (57)Protein biosynthesis22P100817170/226/8YKR059wEukaryotic initiation factor 4A1.60.05509RibosomeiRibosomal proteins are predominantly NE-ER-associated (57)Protein biosynthesis23P256942029/35/9YDL126cCell division cycle protein 48 (Cdc48p)5.80.00002ER-associated, nucleus, cytosolERAD, organelle formation, spindle apparatus24P061066032/89/20YLR303wO-Acetylhomoserine sulfhydrolase (MET17)1.50.00630CytoplasmAmino acid metabolism25P041734148/168/15YCL018w3-Isopropylmalate dehydrogenase1.40.00615CytoplasmAmino acid metabolism26, 27P009245723/—210/14YGR254wEnolase 12.20.00199CytoplasmEnergy metabolism28—30P382197027/16/9YBR025cPutative GTP-binding protein2.40.00803CytoplasmUnknown31Q050167622/—66/9YMR226cPutative oxidoreductase2.80.01019UnknownUnknown32Q124477538/205/8YDR071cHypothetical protein YDR071c3.20.00070UnknownUnknowna SwissProt data base: us.expasy.org/sprot/b PLGS 1.1 scoringc MIPS database: mips.gsf.de/projects/fungid Factor: mutant (cdc48S565G) versus wild-type strain: 1 for accumulation of proteine Mitop2 data base: ihg.gsf.de/mitop2/start.jspf According to Ref. 54Sickmann A. 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Cell Biol. 2002; 14: 412-416Crossref PubMed Scopus (33) Google Scholar) Open table in a new tab Mass spectrometry analysis of the 32 altered protein spots in mitochondria identified 23 unique proteins (Table 1), seven of which were established as mitochondrial proteins. Increased ("enrichment") and decreased ("depletion") amounts of mitochondrial proteins in mitochondrial extracts of cdc48S565G cells were observed (e.g. YGR086c (Fig. 2B, panel 2) and maintenance of mitochondrial function 1 (MMF1, Fig. 2B, panel 1), respectively; for quantification of protein spot alterations see Table 1).The observed depletion of MMF1 (Fig. 2B, panel 1) and ketol acid reductoisomerase (Ilv5p, Table 1), two mitochondrial proteins fundamental for the stability of mitochondrial DNA (32Oxelmark E. Marchini A. Malanchi I. Magherini F. Jaquet L. Hajibagheri M.A. Blight K.J. Jauniaux J.C. Tommasino M. Mol. Cell Biol. 2000; 20: 7784-7797Crossref PubMed Scopus (54) Google Scholar, 33Zelenaya-Troitskaya O. 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