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MPV17L2 Is Required for Ribosome Assembly in Mitochondria Ilaria Dalla Rosa1,†, Romina Durigon1,†, Sarah F

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MPV17L2 Is Required for Ribosome Assembly in Mitochondria Ilaria Dalla Rosa1,†, Romina Durigon1,†, Sarah F 8500–8515 Nucleic Acids Research, 2014, Vol. 42, No. 13 Published online 19 June 2014 doi: 10.1093/nar/gku513 MPV17L2 is required for ribosome assembly in mitochondria Ilaria Dalla Rosa1,†, Romina Durigon1,†, Sarah F. Pearce2,†, Joanna Rorbach2, Elizabeth M.A. Hirst1, Sara Vidoni2, Aurelio Reyes2, Gloria Brea-Calvo2, Michal Minczuk2, Michael W. Woellhaf3, Johannes M. Herrmann3, Martijn A. Huynen4,IanJ.Holt1 and Antonella Spinazzola1,* 1MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK, 2MRC Mitochondrial Biology Unit, Wellcome Trust-MRC Building, Hills Road, Cambridge CB2 0XY, UK, 3Cell Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany and 4Centre for Molecular and Biomolecular Informatics, Radboud University Medical Centre, Geert Grooteplein Zuid 26–28, 6525 GA Nijmegen, Netherlands Received January 15, 2014; Revised May 7, 2014; Accepted May 23, 2014 ABSTRACT INTRODUCTION MPV17 is a mitochondrial protein of unknown func- The mammalian mitochondrial proteome comprises 1500 tion, and mutations in MPV17 are associated with or more gene products. The deoxyribonucleic acid (DNA) mitochondrial deoxyribonucleic acid (DNA) mainte- inside mitochondria DNA (mtDNA) contributes only 13 ∼ nance disorders. Here we investigated its most sim- of these proteins, and they make up 20% of the subunits ilar relative, MPV17L2, which is also annotated as of the oxidative phosphorylation (OXPHOS) system, which produces much of the cells energy. All the other proteins a mitochondrial protein. Mitochondrial fractionation of the organelle are nuclear encoded, synthesized in the cy- analyses demonstrate MPV17L2 is an integral in- tosol and then imported into the mitochondria. A substan- ner membrane protein, like MPV17. However, un- tial number of these proteins have roles associated with the like MPV17, MPV17L2 is dependent on mitochondrial structure, production and maintenance of the respiratory DNA, as it is absent from ␳0 cells, and co-sediments chain and adenosine triphosphate synthase, being struc- on sucrose gradients with the large subunit of the tural components or assembly factors thereof, or contribu- mitochondrial ribosome and the monosome. Gene tors to mtDNA maintenance and expression. However, the silencing of MPV17L2 results in marked decreases in precise role of many mitochondrial proteins remains un- the monosome and both subunits of the mitochon- known, limiting our understanding of the organelle’s role drial ribosome, leading to impaired protein synthe- in physiological and disease processes. sis in the mitochondria. Depletion of MPV17L2 also The construction of a mitochondrial proteome database comprising over 1000 proteins has facilitated the discov- induces mitochondrial DNA aggregation. The DNA ery of mitochondrial disease-genes, such as MPV17 (1). In and ribosome phenotypes are linked, as in the ab- 2006, the MPV17 protein, previously assigned as having sence of MPV17L2 proteins of the small subunit of peroxisomal localization (2), was predicted instead to be a the mitochondrial ribosome are trapped in the en- mitochondrial protein (3) and then experimentally shown to larged nucleoids, in contrast to a component of the localize exclusively to the inner membrane of mitochondria large subunit. These findings suggest MPV17L2 con- (3). In the latter study, MPV17 dysfunction was also linked tributes to the biogenesis of the mitochondrial ribo- to a form of mitochondrial DNA depletion syndrome (3), some, uniting the two subunits to create the transla- and later with multiple deletions of mtDNA (4,5). However, tionally competent monosome, and provide evidence neither the function of the MPV17 protein, nor the mecha- that assembly of the small subunit of the mitochon- nism leading to mtDNA perturbation is currently known. drial ribosome occurs at the nucleoid. In mammals, MPV17 is homologous to three other pro- teins: MPV17-like protein (MPV17L), MPV17-like 2 pro- tein (MPV17L2 or FKSG24) and peroxisomal membrane protein 2 (PXMP2). Existing literature suggest a peroxi- somal localization for PXMP2p (6,7) and dual localiza- *To whom correspondence should be addressed. Tel: +44 20 8816 2273; Fax: +44 20 8906 4477; Email: [email protected]. †The authors wish it to be known that, in their opinion, the first three authors should be regarded as Joint First Authors. C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2014, Vol. 42, No. 13 8501 tion of MPV17L in mitochondria and peroxisomes (8,9). 5-CUGCACU ACUGGUACUUGU-3 A3: s39422, 5- A recent study proposes that PXMP2 forms a constitu- CCCATGAAGATGGATGATCA-3, or Origene O1: 5- tively open pore within the peroxisomal membrane, which UGAUCAUCCAUCUUCAUGG-3) or an unrelated non- is voltage-independent and displays weak cationic selectiv- targeting siRNA (Ambion Silencer select siRNA negative ity (10). Hitherto, nothing was known about the function of control 4390846) at 10 nM delivered using Lipofectamine MPV17L2. However, previous studies of the mitochondrial RNAiMax (Life Technologies) according to manufacturer’s proteome have assigned it as a mitochondrial protein based specifications. HeLa cells underwent either one ortwo on Bayesian integration of genomics data (1) and a green rounds of siRNA treatment, with the second transfection fluorescent protein (GFP) tagged version of the protein is performed 72 h after the initial transfection. After 6 days, targeted to the mitochondria (11). cells were harvested and processed for quantitative real- Here we resolve the phylogenetic relationships of the time PCR (Q-PCR), immunoblotting, sucrose gradient, im- four mammalian MPV17-related proteins, and report a first munofluorescence or mitochondrial translation analysis. characterization of the homologue most similar to MPV17, namely MPV17L2. We show that MPV17L2 is an inner Immunofluorescence and electron microscopy mitochondrial membrane protein that is associated with mitochondrial nucleic acids. Specifically, MPV17L2 inter- HeLa cells grown on glass coverslips were treated with acts with the large subunit of the mitochondrial ribosome 100 nM MitoTrackerR Red for 30 min, fixed with 4% and the monosome, and when its expression is reduced by paraformaldehyde in phosphate-buffered saline (PBS) and ribonucleic acid (RNA) interference, the ribosome is dis- permeabilized with 0.3% Triton X-100 in PBS contain- rupted and translation in the mitochondria is impaired, in- ing 5% FBS. After permeabilization, samples were blocked dicating MPV17L2 plays an important role in ribosomal with 5% FBS in PBS and incubated with the indicated biogenesis in the organelle. primary antibodies: anti-DNA (dilution 1:150; Progen), MRPS27 (dilution 1:200, Proteintech), MRPS18 (dilution MATERIALS AND METHODS 1:200, Proteintech), MRPL45 (dilution 1:200, Proteintech). After three washes, slides were incubated with the fol- Plasmid preparation lowing secondary antibody: AlexaFluor R -488 goat–anti- Human complementary DNA (cDNA) specifying MPV17 mouse antibodies or AlexaFluorR 568 donkey anti-rabbit (IMAGE: 5217853) was introduced into Flp-In T-REx hu- (dilution: 1:1000; Molecular Probes). Finally, cover slips man embryonic kidney cells (HEK293T, Life Technologies) were washed with PBS and mounted on glass slides by in- to establish inducible, transgenic cell lines. The transgene version over ProLongR Gold Antifade Reagent that in- carried a carboxy-terminal linker sequence followed by oc- cludes 4’,6-diamidino-2-phenylindole (DAPI) nuclear stain. tapeptide (DYKDDDDK) (FLAG) and StrepII tags. In preparation for electron microscopy HeLa cells were fixed in 2% glutaraldehyde/2% paraformaldehyde for 30 Cell culture and transfection min, and then incubated for 1 h with 1% osmium tetroxide using 0.1 M sodium cacodylate buffer pH 7.2, at room tem- HEK293T cells were grown in Dulbecco’s Modified Ea- perature. The samples were then dehydrated and embedded gle’s Medium (Life Technologies) supplemented with 10% in Epon resin. Sections were stained with ethanolic uranyl fetal bovine serum (Fetal bovine serum (FBS), Hyclone) acetate and Reynold’s lead citrate, and viewed with a Jeol 1% penicillin and streptomycin (PS, Life Technologies), 15 100EX Transmission electron microscopy (TEM). Images ␮g/ml BlasticidinS and 100 ␮g/ml Zeocin (Biosciences). were captured with a Gatan Orius 1000 Charge-coupled de- For the generation of inducible transgenic MPV17 FLAG- vices (CCD). StrepII cell lines, transfection was mediated using Lipo- fectamine 2000 (Life Technologies) according to manufac- turer’s guidelines. Following transfection, cells underwent Mitochondrial isolation, protease protection assay, carbonate selection in DMEM supplemented with 10% tetracycline- extraction and iodixanol gradient fractionation free FBS (Biochrom), 1% PS, 15 ␮g/ml BlasticidinS and 100 Mitochondria were isolated as described (13). Briefly, ␮g/ml hygromycinB (Sigma). Gene expression was induced HEK293T cells were disrupted by homogenization in hy- by adding doxycycline (Sigma) to the culture medium with a potonic buffer (20 mM HEPES pH 8, 5 mM KCl, 1.5 final concentration of 10/ ng ml for 24 h. HEK293T ␳ 0 cells mM MgCl and 2 mM DTT), and mixed with a mannitol– were generated
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