Atypical Rho GTPases Have Roles in Mitochondrial Homeostasis and Apoptosis

The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Here we report the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrialRho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae,Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early during evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induced an aggregation of the mitochondrial network and resulted in an increased apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis. The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Here we report the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrialRho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae,Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early during evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induced an aggregation of the mitochondrial network and resulted in an increased apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis. Small GTPases have been shown to be pivotal signaling intermediates in cell growth, cell cycle progression, cell survival, cell transformation, and cell trafficking (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 2Hall A. Science. 1998; 279: 509-514Google Scholar, 3Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Google Scholar). The potential of small GTPases to function as signaling switches resides in their ability to cycle between active, GTP-bound states, and inactive, GDP-bound states. This cycling is regulated in a precise manner by guanine nucleotide exchange factors (4Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Google Scholar), GTPase-activating proteins, and guanine nucleotide dissociation inhibitors (GDIs) 1The abbreviations used are: GDI, guanine nucleotide dissociation inhibitor; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; JNK/SAPK, Jun N-terminal kinase/stress-activated protein kinase; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4′,6-diamidino-2-phenyindole; FCS, fetal calf serum; PBS, phosphate-buffered saline; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone 1The abbreviations used are: GDI, guanine nucleotide dissociation inhibitor; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; JNK/SAPK, Jun N-terminal kinase/stress-activated protein kinase; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4′,6-diamidino-2-phenyindole; FCS, fetal calf serum; PBS, phosphate-buffered saline; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone (5Zalcman G. Dorseuil O. Garcia-Ranea J.A. Gacon G. Camonis J. Mol. Subcell. Biol. 1999; 22: 85-113Google Scholar, 6Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1998; 245: 641-645Google Scholar). Guanine nucleotide exchange factors stimulate the replacement of GDP by GTP, whereas GTPase-activating proteins stimulate the intrinsic GTP hydrolysis of the GTPase. GDIs act by blocking GDP dissociation, and in resting cells, Rho GTPases are thought to reside in an inactive complex with RhoGDI. According to this model, cell stimulation leads to a dissociation of the complex, resulting in the subsequent activation of the released Rho GTPase (6Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1998; 245: 641-645Google Scholar). The proto-oncogene ras was the first small GTPase to be identified over 20 years ago (see Ref. 7Shields J.M. Pruitt K. McFall A. Shaub A. Der C.H. Trends Cell Biol. 2000; 10: 147-154Google Scholar and references therein); however, the recently published draft of the human genome indicate the presence of roughly 150 genes encoding small GTPases (8Venter C.J. et al.Science. 2001; 291: 1304-1351Google Scholar, 9International Human Genome Sequencing Consortium Nature. 2001; 409: 860-921Google Scholar). Among these genes, the rho subfamily has been shown to be involved specifically in regulating the morphogenic and motile properties of vertebrate cells, primarily by affecting the actin filament system (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 2Hall A. Science. 1998; 279: 509-514Google Scholar, 3Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Google Scholar). Most of our current knowledge regarding the Rho GTPases originates from work on the triad RhoA, Rac1, and Cdc42, which have each been found to regulate distinct actin-containing structures. RhoA regulates the formation of focal adhesions and the subsequent assembly of stress fibers (10Ridley A.J. Hall A. Cell. 1992; 70: 389-399Google Scholar); Rac1 regulates the formation of membrane lamellae or lamellipodia (11Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Google Scholar), and Cdc42 triggers the outgrowth of peripheral spike-like protrusions, also known as filopodia (12Nobes C.D. Hall A. Cell. 1995; 81: 53-62Google Scholar). However, over the years, it has become evident that the Rho GTPases are involved in the regulation of several additional cellular processes. Rac1 and Cdc42 participate in transcriptional control via the Jun N-terminal kinase/stress-activated protein kinase and p38MAPK signaling cascades; RhoA has a role in serum-response factor-regulated gene transcription, and all three contribute to transcriptional activation via NFκB signaling pathway (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 2Hall A. Science. 1998; 279: 509-514Google Scholar, 3Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Google Scholar). Furthermore, the Rho GTPases are also participants in signaling pathways that control cell cycle progression and apoptosis (13Aznar S. Lacal J.C. Cancer Lett. 2001; 165: 1-10Google Scholar). So far, 20 distinct genes encoding family members of Rho GTPases have been identified. These genes can be further divided into seven subgroups: cdc42 (consisting of cdc42, tc10, tcl, chp, and wrch1/chp2), rac (rac1, rac2, rac3, and rhoG), rho (rhoA, rhoB,and rhoC), rnd (rnd1, rnd2, andrnd3), rhoD (rhoD and rif), and rhoH (see Ref. 14Aspenström P. Exp. Cell Res. 1999; 246: 20-25Google Scholar and references therein and Refs.15Vignal E. De Toledo M. Comunale F. Landopoulou A. Gauthier-Rouvière C. Blagny A. Fort P. J. Biol. Chem. 2000; 275: 36457-36464Google Scholar, 16Ellis S. Mellor H. Curr. Biol. 2000; 10: 1387-1390Google Scholar, 17Tao W. Pennica D. Xu L. Kalejta R.F. Levine A.J. Genes Dev. 2001; 15: 1796-1807Google Scholar). The seventh subgroup is formed by three additionalrho-like genes, called kiaa0740, kiaa0717, andkiaa0878, which have been identified by the Kazusa Institute in Japan (18Nagase T. Ishikawa K. Suyama M. Kikuno R. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 355-364Google Scholar). These genes encode human orthologs of theDrosophila melanogaster rhoBTB gene, suggesting that more appropriate names for them would be rhoBTB1, rhoBTB2, and rhoBTB3, respectively (18Nagase T. Ishikawa K. Suyama M. Kikuno R. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 355-364Google Scholar, 19Rivero F. Dislich H. Glöckner G. Noegel A.A. Nucleic Acids Res. 2001; 29: 1068-1079Google Scholar). The work in this article describes a novel subgroup of the Rho GTPases. This subgroup is formed by two Rho-like genes, which were named Miro (for mitochondrial Rho). These genes encode proteins that are similar to the Rho GTPases in their N-terminal GTPase domains, but in addition they contain potential calcium-binding motifs, so called EF-hands (20Kawasaki H. Nakayama S. Kretsinger R.H. Biometals. 1998; 11: 277-295Google Scholar, 21Nelson M.R. Chazin W.J. Biometals. 1998; 11: 297-318Google Scholar), and an additional putative GTPase domain in the C terminus which is diverged from the Rho GTPases. Data presented in this report showed that ectopically expressed Miro-1 and Miro-2, as well as endogenous Miro-1, are present at mitochondria. Furthermore, transient transfection of a constitutively active Miro-1 (Miro-1/Val-13) destroyed the mitochondrial network, which collapsed into perinuclear assemblies; moreover, it resulted in an increased apoptosis rate. These data suggested a role for Miro proteins in mitochondrial homeostasis and in apoptosis. Miro-1 and Miro-2 were identified by searching the public DNA and protein data bases for novel members of the Rho GTPases employing the Blast search motor. The translated amino acid sequence for Miro-1 and Miro-2 were then used to search EST data bases for the presence of EST clones encoding putative full-length Miro-1 and Miro-2, and two such clones were obtained from the UK Human Genome Mapping Project Resource Centre in Hinxton, Cambridge, UK. The nucleotide sequences of Miro-1 and -2 have been deposited in the GenBankTM data base with accession numbers AJ517412(Miro-1) and AJ517413 (Miro-2). DNA fragments encoding full-length Miro-1 and Miro-2 were generated by PCR and subcloned into the pRK5Myc vector. The Miro-1/Val-13 and Miro-1/Asn-18 mutants were generated employing the QuickChange protocol (Stratagene) according to the procedure provided by the manufacturer. The fidelity of all DNA constructs was conformed by DNA sequencing employing the ABI Prism 310 Genetic Analyzer. cDNA probes of the open reading frame of Miro-1 and Miro-2, respectively, were labeled with 32PdCTP employing the rediprime labeling kit (AmershamBiosciences). The probes were thereafter hybridized to hybridization-ready Northern blots (Human Multiple Tissue Northern blot, Clontech) according to the ExpressHyb (Clontech) protocol provided by the manufacturer. NIH3T3 cells and COS 7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS and penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2. For immunostaining purposes, cells were seeded on coverslips and transfected by LipofectAMINE or LipofectAMINE Plus (Invitrogen) according to the protocols provided by the manufacturer. Transfections of COS 7 cells for immunoprecipitation purposes were performed by the DEAE-dextran method essentially as described before (22Richnau N. Aspenström P. J. Biol. Chem. 2001; 276: 35060-35070Google Scholar). Metabolic 35S labeling of Miro-1 was performed as described before (22Richnau N. Aspenström P. J. Biol. Chem. 2001; 276: 35060-35070Google Scholar). Miro-1 immunoprecipitates were subjected to SDS-PAGE, and the gels were fixed for 15 min in 25% methanol and 7.5% acetic acid. The gels were thereafter dried and exposed on a PhosphorImager (Fujix BAS 2000). A Miro-1-specific antibody was produced by immunizing rabbits with a keyhole limpet hemocyanin-conjugated Miro-1-specific peptide representing amino acid residues 560–574 of human Miro-1. Mouse monoclonal anti-Myc (9E10) and rabbit polyclonal anti-Myc antibodies (Santa Cruz Biotechnology) were used to determine the subcellular localization of Myc-tagged Miro-1 and Miro-2 as well as Miro-1 mutants. The mouse monoclonal M30 antibody (Roche Molecular Biochemicals) was used to stain apoptotic cells. MitoTracker Green FM (Molecular Probes) and a mouse monoclonal anti-cytochrome c antibody (Pharmingen) were used according to the protocol supplied by the manufacturer to visualize mitochondria, and 4′,6-diamidino-2-phenyindole (DAPI) was used to visualize nuclei. Filamentous actin was visualized by TRITC-conjugated phalloidin (Sigma). Microtubules were visualized by a mouse monoclonal anti-α-tubulin antibody (Sigma). The following secondary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-mouse, FITC-conjugated anti-rabbit, TRITC-conjugated anti-rabbit (Dako), and a TRITC-conjugated anti-mouse (Jackson ImmunoResearch). The caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK were obtained from Calbiochem. For the immunocytochemistry, transiently transfected NIH3T3 and COS 7 cells were grown on coverslips and fixed in 3% paraformaldehyde in PBS for 20 min at 37 °C. The cells were washed with PBS and permeabilized in 0.2% Triton X-100 in PBS for 5 min. The cells were thereafter washed again and incubated in the presence of 5% FCS in PBS for 30 min. Primary as well as secondary antibodies were diluted in PBS containing 5% FCS. Cells were incubated with primary antibodies followed by secondary antibodies for intervals of 1 h with a washing step in between. The coverslips were in on Cells were by a employing the a in the public genomic DNA sequence data bases generated by the human genome the presence of several novel members of the Rho GTPases (8Venter C.J. et al.Science. 2001; 291: 1304-1351Google Human Genome Sequencing Consortium Nature. 2001; 409: 860-921Google Scholar). sequence encoding two putative Rho were that these clones encoded two Rho which were named Miro-1 and Miro-2, Miro-1 and Miro-2 were found to be to each and proteins encoded of amino acid residues with of A and The amino acid sequence of Miro-1 and -2 revealed an domain the N-terminal encoded a GTPase which is to the Rho GTPases 1 This domain was followed by two EF 1 a of domain that to (20Kawasaki H. Nakayama S. Kretsinger R.H. Biometals. 1998; 11: 277-295Google Scholar, 21Nelson M.R. Chazin W.J. Biometals. 1998; 11: 297-318Google Scholar). a potential GTP-binding but in this to the Rho was found in the C terminus 1 Miro a a domain found in the C of small GTPases. The a that membrane of the The domain of Miro that the proteins are regulated in a manner distinct from the Rho of domain of human Miro-1 and of the amino acid sequences of human Miro-1 and amino acid residues in Miro-1 and Miro-2, and amino acid were performed by the of GTPase domain of Miro-1 and Miro-2 with human Rac1, RhoA, and the amino acid residues and of Rho GTPases. acid in of these in GTP hydrolysis of the GTPase. Miro-1 has and residues in these whereas Miro-2 has residues in of the EF of Miro the calcium-binding for EF (20Kawasaki H. Nakayama S. Kretsinger R.H. Biometals. 1998; 11: 277-295Google Scholar, 21Nelson M.R. Chazin W.J. Biometals. 1998; 11: 297-318Google of the GTPase domain of Miro proteins from eukaryotic of the of all known Miro Northern of human Miro-1 and Miro-2 in the human in the Interestingly, data base the presence of genes, to in several eukaryotic organisms elegans, D. melanogaster 1 This that the Miro genes evolved early during evolution. Northern employing human Northern the presence of Miro in human 1 Miro-1 of a of was in and and Miro-2 of was in and 1 The Rho GTPases have been shown to have a on the of the actin (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 2Hall A. Science. 1998; 279: 509-514Google Scholar, 3Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Google Scholar). For this the on the by of Miro-1 and -2 was encoding Myc-tagged Miro-1 and Miro-2 were transiently transfected into NIH3T3 cells which Miro was visualized by a whereas actin was visualized with TRITC-conjugated Microtubules were visualized by an antibody Miro-1 Miro-2 the of the actin filament system A or the Interestingly, it was that Miro followed a distinct Miro-1 and Miro-2 were in in a manner of the mitochondrial network A Miro expressing the N-terminal GTPase domain into such this mutant was in the of transfected cells to the localization of Miro with mitochondria, NIH3T3 cells were transiently transfected with and with the MitoTracker Green Miro-1 to a large with the MitoTracker suggesting that Miro-1 is present at the transfected Miro-2 with mitochondrial in a similar with the protein further the of Miro as a protein A peptide from the C terminus of Miro-1 was and used to a Miro-1-specific in The was over a of Miro-1-specific The as well as the was in immunoprecipitation employing COS 7 cells transiently transfected with The Miro-1 and the as well as the a of This was by with the Miro-1 with the Miro-1-specific peptide a number of cell were for of Miro-1 by Human cells and human cells of Miro-1, whereas NIH3T3 and COS 7 cells expressed of Miro-1 and cells in a that of their For this the of Miro-1, COS 7 cells to be more for of the mitochondrial The Miro-1-specific antibody was used to stain COS 7 cells in to endogenous protein was to mitochondria. The to a large with moreover, the Miro-1-specific was by with the blocking these suggested that endogenous Miro-1 is a mitochondrial protein employing active and mutants of Rho GTPases have been used to the of these proteins in pathways (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Google Scholar, 3Ridley A.J. Trends Cell Biol. 2001; 11: 471-477Google Scholar). with the for of RhoA, Rac1, and Cdc42, we a active mutant Miro-1 in which the amino acid at was with a This the GTPase in a GTP-bound the a Miro-1 was by to an a that the GTPase in a GDP-bound or was transiently transfected into NIH3T3 cells or into COS 7 cells. The overexpression of Miro mutants on the cell or on the of the actin filament system or the Interestingly, the Miro-1/Val-13 was in a manner with The cells the mitochondrial network, and Miro-1/Val-13 was into large perinuclear in NIH3T3 and COS 7 cells with MitoTracker Green FM that the mitochondrial network in collapsed in these cells, and the were in these perinuclear expressing cells and a of the cells a mitochondrial however, cells with were also these These data suggested that the of Miro-1 was for the of the mitochondrial network and the mitochondrial c was present in the collapsed mitochondrial c has been shown to be released from the during apoptosis J.C. Science. 1998; Scholar); COS 7 cells were transiently transfected with or and for and localization and h COS 7 cells the with the to the transfected NIH3T3 This was further by which a between c and Miro-1 overexpression resulted again in a of the mitochondrial Miro-1/Val-13 was present in the mitochondrial but it was also into the The c was present in the mitochondrial but an increased number of cells with c were h of mitochondrial homeostasis have been shown to in an increased rate of apoptosis D. J.C. Scholar). a of cells over with the of cells, which an increased rate of apoptosis in this cell For this we stained COS 7 cells transiently transfected with or with a antibody as well as with the apoptosis antibody M30 specifically the the of M30 cells that was also transfected with the Miro-1 mutants. A in M30 cells was in the of cells expressing h the M30 and cells showed a network of However, a the M30 was to in in the cells 5 The Miro-1/Val-13 and M30 cells also which be visualized by 5 the between M30 cells and cells. the of Miro-1/Val-13 of the cells were in cells expressing Miro-1 or or respectively, of the cells were apoptosis 5 The Miro-1/Val-13 induced apoptosis was from to 7.5% the cells were with the caspase Z-VAD-FMK of the cells with Z-DEVD-FMK also the from to 5 indicating that this is on the caspase these data that Miro proteins are mitochondrial and that the of Miro the homeostasis of to an increased rate of apoptosis. The of Cdc42, or Rho in cell and are well A of the Rho GTPases are to this however, are increased that Rho GTPases have cellular For members of the subfamily have been suggested to function as to Rho in to their activation leads to stress fibers and a to the a of cells C.D. S. Hall A. P. J. Cell Biol. 1998; Scholar). has been to activation of the p38MAPK and NFκB signaling pathways H. Mol. Cell. Biol. 22: Scholar). Furthermore, and to have during membrane but they also the formation of by a distinct from Cdc42 or the GTPases S. Mellor H. Curr. Biol. 2000; 10: 1387-1390Google Scholar, C. R. M. H. M. P. A. M. Nature. Scholar). and are two Cdc42 family GTPases that are involved in the formation of peripheral such as lamellipodia and filopodia W. Pennica D. Xu L. Kalejta R.F. Levine A.J. Genes Dev. 2001; 15: 1796-1807Google Scholar, A. A. A. A. Curr. Biol. 1998; Scholar). these the for a model, which that of cellular a regulated activation of Rho GTPases. Miro proteins are by of their they are the small but also by the presence of additional protein domain structures. of atypical Rho-like proteins, the has also a domain that is from the Rho GTPases F. Dislich H. Glöckner G. Noegel A.A. Nucleic Acids Res. 2001; 29: 1068-1079Google Scholar). The in addition to their N-terminal GTPase domains, two complex, and domains T. A.J. Mol. Cell. Biol. 2001; Scholar). This of domain was first identified in a number gene but were found in several proteins from to this domain is found in proteins with in the regulation of and however, the cellular function for F. Dislich H. Glöckner G. Noegel A.A. Nucleic Acids Res. 2001; 29: 1068-1079Google Scholar). the of our of the Rho GTPases have been in mitochondrial and in this Miro a subfamily of Rho GTPases with Interestingly, of proteins, to the Rho GTPases but with GTP-binding has been shown to have in mitochondrial in D. melanogaster and C. elegans, as well as in cells has identified three GTPases in this and Scholar, A. J. Cell Biol. 2000; Scholar). is a protein on the of it regulates of the mitochondrial membrane during mitochondrial T. A.J. Mol. Cell. Biol. 2001; Scholar). The is regulated by This protein to and regulates mitochondrial membrane A. J. Cell Biol. 2000; Scholar). to regulate several of mitochondrial membrane Interestingly, overexpression of a constitutively active mutant of the in COS 7 cells resulted in the formation of a mitochondrial network, by a in the of the mitochondrial an over of mitochondrial A. J. Cell Sci. 2001; Scholar). This to the mitochondrial in cells expressing constitutively active these be by an in mitochondrial membrane however, the for to be homeostasis is a cellular and in the that mitochondrial function triggers the apoptosis J.C. Science. 1998; Scholar). this the are as cellular the of factors such as and c into the which in the caspase J.C. Science. 1998; Scholar, D. J.C. Scholar). Rho GTPases have been in of apoptosis in Ref. S. Lacal J.C. Cancer Lett. 2001; 165: 1-10Google Scholar). For Cdc42 is for the apoptosis of cells Sci. S. A. 1998; Scholar). Cdc42 is a caspase and of the caspase in a apoptosis S. J. Biol. Chem. 2001; 276: Scholar). of the constitutively active mutant Miro-1/Val-13 in COS 7 cells increased the apoptotic rate of expressing cells. The for this to be but be the mitochondrial as the aggregation of the mitochondrial Interestingly, it has been shown that of the of in such a that the mitochondrial was resulted in apoptosis S. F. C.L. Dev. Cell. 2001; Scholar), further the for a between mitochondrial and for cell have identified an family of Rho with atypical domain and cellular employing NIH3T3 and COS 7 cells that Miro is a of and that the protein is involved in the regulation of mitochondrial homeostasis and apoptosis.