A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells
© Weiher et al. 2016
Received: 17 July 2015
Accepted: 16 February 2016
Published: 27 February 2016
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.
KeywordsMPV17 monoclonal antibody Mitochondrial DNA depletion syndrome Lysosomes Endosomes Mitochondria Peroxisomes
Mutations in the human MPV17 gene have been firstly discovered to be causal for the lethal liver disease Mitochondrial DNA Depletion Syndrome (MDDS) by Spinazzola et al. in 2006 . Since then various additional mutations within this gene were described to cause the syndrome [5, 8, 23]. In contrast to other proteins of which gene mutations cause MDDS such as POLG, TK2, or DGOUK , this protein is not obviously involved in replication or in nucleic acid metabolism, but appears to be a membrane protein of unknown molecular function [21, 25, 26]. MPV17 gene knockout in mice has been described earlier as causal of glomerulosclerosis [2, 14, 25] and inner ear disease , reminiscent of chemical damage by Adriamycin . Although these mice also showed mitochondrial DNA depletion, they displayed no major liver phenotype . A functional link between the PRKDC repair protein, the mouse kidney phenotype as well as the mitochondrial depletion phenotype has been established by Papeta et al. . Human MPV17 protein expression can rescue the phenotype in transgenic mice, negative for murine MPV17 . A functional homolog of the MPV17 protein was identified in yeast (Sacharomyces cerevisiae) and the human gene can rescue the yeast phenotype in Sym1 negative yeast as well . In addition, a mutant in the zebra fish (Dario rerio) MPV17 homolog tra has been described . The yeast and fish mutant phenotypes appear remarkably different from the mutant phenotypes in mammals: Sym 1 negative yeast fails to grow at elevated temperature in ethanol , while tra negative fish are viable but show a transparent appearance . Finally, MPV17 is a member of a protein family, including MPV17 like proteins [6, 19] and the peroxisomal membrane protein PXMP22 [7, 18]. The hypothesis that MPV17 protein might constitute a channel allowing—in vertebrates—nucleotides or—in yeast—metabolic intermediates to pass though internal membranes was recently reviewed .
The publication describing the causative role of MPV17 mutations in the human liver disease MDDS included an analysis of the intracellular localisation of the MPV17 protein in human cells based mainly on studies of transfected cells over-expressing a c-terminally tagged recombinant protein, it was stated that this protein localised to the inner membrane of mitochondria . Supporting this notion, it has been established that the yeast homolog of MPV17, SYM1, indeed localises to this cell compartment [17, 22]. An earlier study , however, had localised the MPV17 gene product to peroxisomes, again based on immunofluorescence data and supported by the similarity of the protein to the bona-fide peroxisomal membrane protein PXMP22 .
To clarify this contradiction we here present co-localisation studies using novel anti-human MPV17 monoclonal mouse antibodies.
Cell lines and tissue culture
Human osteosarcoma cells (U2OS) cells were originally obtain from the American tissue culture collection (ATCC® HTB-96™), kept at the Heinrich Pette Institut for several years and used in . Primary murine embryonic fibroblasts (MEF) were produced from day 11 MPV17 +/+ and MPV17 −/− embryos respectively according to Zwacka et al. . U2OS and mouse MEF cells were cultured under standard conditions. Stable transformants were selected using 1 mg/ml G418 (Life Technologies). Hybridomas were generated as described , and clones were grown in RMPI media including 10–20 % FCS. The clones were monitored for IgG production by immunoblotting using anti-IgG antibodies and the ECL System (Pierce) for detection. Antibodies were purified using a Protein G Sepharose column (Life Technologies) according to the manufacturer’s instructions.
Antibodies and organelle detection
Commercial primary antibodies against MPV17 were: mouse monoclonal anti-human MPV17 antibody (60103-1-Ig, Proteintech), rabbit anti-human MPV17 polyclonal antibody (10310-1-AP, Proteintech); rabbit anti-human MPV17 polyclonal antibody (ab93374, Abcam); goat anti-human MPV17 polyclonal antibody (sc-109551, Santa Cruz), rabbit anti-MPV17 C-terminal region polyclonal antibody ARP73712-P050 Insight Biotechnology), rabbit anti-MPV17 N-terminal region polyclonal antibody AP8749a-ev-AB, BioCat).
Further primary antibodies used were: mouse polyclonal anti-human catalase antibody (Abcam ab88650); mouse monoclonal anti-complex IV mitochondrial subunit I (Invitrogen 459600); rabbit polyclonal anti-human PMP70 antibody (gift from Wilhelm Just, University of Heidelberg); rabbit anti-human Rab 7 antibody (Sigma R4779); goat anti- human cathepsin D antibody (Santa Cruz sc 6486); rabbit anti-human EEA1 antibody (Novus-Biologicals, NB-300-502); goat anti-human LAMP1 antibody (Santa Cruz, sc 8098); mouse monoclonal anti-flag antibody (Sigma, M2, F3165), mouse monoclonal anti-human beta-actin antibody (Sigma, AC74, A5316).
Primary monoclonal Antibodies 5D2 and 6F5 were isolated from mass cultures and purified over protein G Sepharose (Life Technologies). A Rabbit polyclonal anti-human PMP70 antibody was a gift from Wilhelm Just, University of Heidelberg. Further primary antibodies used were: rabbit anti-human Rab 7 antibody (Sigma R4779); goat anti-human cathepsin D antibody (Santa Cruz sc 6486); rabbit anti-human EEA1 antibody (Novus-Biologicals, NB-300-502); goat anti-human LAMP1 antibody (Santa Cruz, sc 8098); mouse monoclonal anti-human beta-actin antibody (Sigma, AC74, A5316).
Secondary antibodies: HRP labeled anti-mouse IgG antibody (Pierce); Alexa Fluor 555 and Alexa Fluor 488 with appropriate anti-mouse, anti-rabbit, and anti-goat specificity were from Life Technologies. For mitochondrial staining, MitoTracker Red 7510 was employed according to the manufacturer’s instructions.
Cell extracts and immunoblotting
An MPV17 expression construct (SC118652) was from origene. For details see: www.origene.com/cdna/.
When tested in immunofluorescence studies on U2OS cells, 6F5 did not display specific reactivity (data not shown), while 5D2 generated a characteristic punctate pattern, which in the Image J analysis of confocal microscopic pictures showed partial colocalisation with the peroxisomal marker PMP70 (polyclonal rabbit PMP70 antibody, gift from W. Just, University of Heidelberg), (Fig. 3 top). However, the pattern did not coincide with mitochondrial staining as displayed by MitoTracker Red (MP07510, Invitrogen) (Fig. 3 bottom). Thus, the staining pattern was not exclusively peroxisomal and not mitochondrial, as had been reported for MPV17 localisation in earlier studies, respectively [21, 26].
In this work we have characterised two monoclonal antibodies raised against a bacterially expressed N-terminal GST fused to the human MPV17 protein. These antibodies, specific for the MPV17 part of the fusion protein recognise a band of the expected size in human U2OS cells. While 6F5 cross reacts with other proteins 5D2 appears to be monospecific. 5D2 creates a punctate pattern in immunofluorescence studies partially colocalising with peroxisomal, early endosomal, and lysosomal markers but not with mitochondria. 5D2 does bind only weakly but specifically to the murine MPV17 homolog (Fig. 1), suggesting that the mouse monoclonal might bind to an epitope where murine and human MPV17 diverge. This could be the c-terminus of the molecule, which is the region of strongest divergence. In line with this idea is the fact that the c-terminus of the MPV17 molecule was exposed in the bacterial n-terminal fusion to GST antigen used for immunisation. Moreover, MPV17 molecules fused at the c-terminus to other proteins cannot be detected by 5D2 (data not shown).
The commercial anti-human MPV17 antibody used in our first approach was a polyclonal rabbit antibody raised against GST-MPV17 fusion protein (abcam ab 93374) and has produced erroneous or ambiguous results (Fig. 2). The rabbit polyclonal antibody from Proteintech used by  leading to mitochondrial localisation was raised against the identical GST-MPV17 fusion and might have been error—prone as well. Furthermore, in addition to possible transfection artifacts, MPV17 and related proteins such as PXMP22 may localise erroneously, when they are fused to detection tags (unpublished and .
MPV17 deficiencies in humans can cause fatal liver disease mediated by mitochondrial DNA depletion in liver cells . However, primary human liver cells were not available for our studies. We therefore had to perform this study on unrelated human tumour cells, and it is possible, that the MPV17 protein might localise differently in different cell types. Yet, preliminary studies on human primary skin fibroblasts show MPV17 colocalisation with LAMP1 as well (unpublished results) and thus corroborate the data on U2OS cells presented above. Thus, our data raise the question of how the mitochondrial MDDS phenotype is generated if the MPV17 gene product is found mainly in other organelles. It has been shown that MPV17 protein forms a membrane channel with a diameter allowing low molecular weight molecules to pass  but the role of the channel is still elusive, particularly considering the different phenotypes that mutations of it can cause in different species . We look forward to provide the antibodies described here to approach these open questions.
HW: Making of constructs, western blots, immunohistochemistry, confocal microscopy. HP and PJD: contribution to the Westernblot in Fig. 1, transfection and immunohistochemistry depicted in Fig. 2. SH and PK: help with growth and purification monoclonal antibodies. SG, MV and JKH: help with and the production of MPV17 negative MEF cells. RMZ: generation of monoclonal antibodies. ES: help with confocal microscopy and Fiji software. KR and HW: technical and financial support; critical reading of the manuscript and most valuable discussion. All authors read and approved the final manuscript.
We thank Dr. Alexander Bartelt and Dr. Katrin Kollmann, UKE Hamburg, for marker antibodies and valuable discussions. This work was supported by the Heinrich-Pette-Institute, Hamburg, Germany; the Institute for Biomedical Aging Research, University of Innsbruck, Austria; the Institutes of Molecular Medicine and Experimental Immunology, Universität Bonn, Germany; and the Department of Biochemistry, Biocenter, University of Oulu, Finland.
The authors declare that they have no competing interests.
All work concerned tissue culture cells for which no specific licences were required. For isolation of the mouse embryonic fibroblasts the use of experimental animals was conducted under the permission of the Animal Experimentation Board of Finland (ESLH-2009-04840/Ym-23).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Antonenkov VD, Isomursu A, Mennerich D, Vapola MH, Weiher H, Kietzmann T, Hiltunen JK. The human mitochondrial DNA depletion syndrome gene MPV17 encodes a non-selective channel that modulates membrane potential. J Biol Chem. 2015;290:3840–61.View ArticleGoogle Scholar
- Binder CJ, Weiher H, Exner M, Kerjaschki D. Glomerular overproduction of oxygen radicals in MPV17 gene-inactivated mice causes podocyte foot process flattening and proteinuria: a model of steroid-resistant nephrosis sensitive to radical scavenger therapy. Am J Pathol. 1999;154:1067–75.PubMed CentralView ArticlePubMedGoogle Scholar
- Brosius U, Dehmel T, Gärtner J. Two different targeting signals direct human peroxisomal membrane protein 22 to peroxisomes. J Biol Chem. 2002;277:774–84.View ArticlePubMedGoogle Scholar
- Copeland WC. Defects in mitochondrial DNA replication and human disease. Crit Rev Biochem Mol Biol. 2012;47:64–74.PubMed CentralView ArticlePubMedGoogle Scholar
- El-Hattab AW, Li F-Y, Schmitt E, Zhang S, Craigen WJ, Wong L-JC. MPV17-associated hepatocerebral mitochondrial DNA depletion syndrome: new patients and novel mutations. Mol Genet Metab. 2010;99:300–8.View ArticlePubMedGoogle Scholar
- Iida R, Yasuda T, Tsubota E, Takatsuka H, Matsuki T, Kishi K. Human MPV17-like protein is localized in peroxisomes and regulates expression of antioxidant enzymes. Biochem Biophys Res Commun. 2006;344:948–54.View ArticlePubMedGoogle Scholar
- Kaldi K, Diestelkötter P, Stenbeck G, Auerbach S, Jäkle U, Mägert HJ, Wieland FT, Just WW. Membrane topology of the 22 kDa integral peroxisomal membrane protein. FEBS Lett. 1993;315:217–22.View ArticlePubMedGoogle Scholar
- Karadimas CL, Vu TH, Holve SA, Chronopoulou P, Quinzii C, Johnsen SD, Kurth J, Eggers E, Palenzuela L, Tanji K, Bonilla E, De Vivo DC, DiMauro S, Hirano M. Navajo neurohepatopathy is caused by a mutation in the MPV17 gene. Am J Hum Genet. 2006;79:544–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Karasawa M, Zwacka RM, Reuter A, Fink T, Hsieh CL, Lichter P, Francke U, Weiher H. The human homolog of the glomerulosclerosis gene MPV17: structure and genomic organization. Hum Mol Genet. 1993;2:1829–34.View ArticlePubMedGoogle Scholar
- Kinkley S, Staege H, Mohrmann G, Rohaly G, Schaub T, Kremmer E, Winterpacht A, Will H. SPOC1: a novel PHD-containing protein modulating chromatin structure and mitotic chromosome condensation. J Cell Sci. 2009;122:2946–56.View ArticlePubMedGoogle Scholar
- Meyer zum Gottesberge AM, Reuter A, Weiher H. Inner ear defect similar to alport’s syndrome in the glomerulosclerosis mouse model MPV17. Eur Arch Otorhinolaryngol. 1996;253:470–4.View ArticlePubMedGoogle Scholar
- Krauss J, Astrinides P, Frohnhöfer HG, Walderich B, Nüsslein-Volhard C. Transparent, a gene affecting stripe formation in Zebrafish, encodes the mitochondrial protein MPV17 that is required for iridophore survival. Biology Open. 2013;2:703–10.PubMed CentralView ArticlePubMedGoogle Scholar
- Löllgen S, Weiher H. The role of the MPV17 protein mutations of which cause mitochondrial DNA depletion syndrome (MDDS): lessons from homologs in different species. Biol Chem. 2015;396:13–25. doi:https://doi.org/10.1515/hsz-2014-0198.View ArticlePubMedGoogle Scholar
- O’Bryan T, Weiher H, Rennke HG, Kren S, Hostetter TH. Course of renal injury in the MPV17-deficient transgenic mouse. J Am Soc Nephrol. 2000;11:1067–74.PubMedGoogle Scholar
- Papeta N, Zheng Z, Schon EA, Brosel S, Altintas MM, Nasr SH, Reiser J, D’Agati VD, Gharavi AG. Prkdc participates in mitochondrial genome maintenance and prevents adriamycin-induced nephropathy in mice. J Clin Invest. 2010;120:4055–64.PubMed CentralView ArticlePubMedGoogle Scholar
- Pircher H, Straganz GD, Ehehalt D, Morrow G, Tanguay RM, Jansen-Dürr P. Identification of human fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) as a novel mitochondrial acylpyruvase. J Biol Chem. 2011;286:36500–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Reinhold R, Krüger V, Meinecke M, Schulz C, Schmidt B, Grunau SD, Guiard B, Wiedemann N, van der Laan M, Wagner R, Rehling P, Dudek J. The channel-forming SYM1 protein is transported by the TIM23 complex in a presequence-independent manner. Mol Cell Biol. 2012;32:5009–21.PubMed CentralView ArticlePubMedGoogle Scholar
- Rokka A, Antonenkov VD, Soininen R, Immonen HL, Pirilä PL, Bergmann U, Sormunen RT, Weckström M, Benz R, Hiltunen JK. Pxmp2 is a channel-forming protein in mammalian peroxisomal membrane. PLoS One. 2009;4:e5090.PubMed CentralView ArticlePubMedGoogle Scholar
- Rosa ID, Durigon R, Pearce SF, Rorbach J, Hirst EM, Vidoni S, Reyes A, Brea-Calvo G, Minczuk M, Woellhaf MW, Herrmann JM, Huynen MA, Holt IJ, Spinazzola A. MPV17L2 is required for ribosome assembly in mitochondria. Nucleic Acids Res. 2014;42:8500–15.PubMed CentralView ArticlePubMedGoogle Scholar
- Schenkel J, Zwacka RM, Rutenberg C, Reuter A, Waldherr R, Weiher H. Functional rescue of the glomerulosclerosis phenotype in MPV17 mice by transgenesis with the human MPV17 homologue. Kidney Int. 1995;48(1):80–4.View ArticlePubMedGoogle Scholar
- Spinazzola A, Viscomi C, Fernandez-Vizarra E, Carrara F, D’Adamo P, Calvo S, Marsano RM, Donnini C, Weiher H, Strisciuglio P, Parini R, Sarzi E, Chan A, DiMauro S, Rötig A, Gasparini P, Ferrero I, Mootha VK, Tiranti V, Zeviani M. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat Genet. 2006;38:570–5.View ArticlePubMedGoogle Scholar
- Trott A, Morano KA. SYM1 is the stress-induced saccharomyces cerevisiae ortholog of the mammalian kidney disease gene MPV17 and is required for ethanol metabolism and tolerance during heat shock. Eukaryot Cell. 2004;3:620–31.PubMed CentralView ArticlePubMedGoogle Scholar
- Uusimaa J, Evans J, Smith C, Butterworth A, Craig K, Ashley N, Liao C, Carver J, Diot A, Macleod L, Hargreaves I, Al-Hussaini A, Faqeih E, Asery A, Al Balwi M, Eyaid W, Al-Sunaid A, Kelly D, van Mourik I, Ball S, Jarvis J, Mulay A, Hadzic N, Samyn M, Baker A, Rahman S, Stewart H, Morris AAM, Seller A, Fratter C, Taylor RW, Poulton J. Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene. Eur J Hum Genet. 2013;22(2):184–91.PubMed CentralView ArticlePubMedGoogle Scholar
- Viscomi C, Spinazzola A, Maggioni M, Fernandez-Vizarra E, Massa V, Pagano C, Vettor R, Mora M, Zeviani M. Early-onset liver mtDNA depletion and late-onset proteinuric nephropathy in MPV17 knockout mice. Hum Mol Genet. 2009;18:12–26.PubMed CentralView ArticlePubMedGoogle Scholar
- Weiher H, Noda T, Gray DA, Sharpe AH, Jaenisch R. Transgenic mouse model of kidney disease: insertional inactivation of ubiquitously expressed gene leads to nephrotic syndrome. Cell. 1990;62:425–34.View ArticlePubMedGoogle Scholar
- Zwacka RM, Reuter A, Pfaff E, Moll J, Gorgas K, Karasawa M, Weiher H. The glomerulosclerosis gene MPV17 encodes a peroxisomal protein producing reactive oxygen species. EMBO J. 1994;13:5129–34.PubMed CentralPubMedGoogle Scholar