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Address correspondence to Jodi Nunnari, Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Ave., Davis, CA 95616. Tel.: (530) 754-9774. Fax: (530) 752-7522. email: jmnunnari{at}ucdavis.edu
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Abstract |
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Key Words: mitochondria; mtDNA; nucleoid; replisome; membrane-spanning
Abbreviations used in this paper: mtDNA, mitochondrial DNA; TAC, tripartite attachment complex; TMS, two membranespanning structure.
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Introduction |
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Proteomic and genetic approaches have identified molecules directly associated with mtDNA within nucleoid structures. These include the mitochondrial-specific DNA-binding proteins Mip1, Abf2, and Mgm101 (Meeusen et al., 1999; Kaufman et al., 2000). Mip1 is a pol- DNA polymerase that possesses 3'-5' exonuclease proofreading activity and represents the only known yeast mtDNA polymerase (Foury, 1989). Abf2 is a relatively abundant HMG-like DNA-binding protein and is thought to function in mtDNA packaging and recombination (Diffley and Stillman, 1991, 1992). Mgm101 is a novel DNA-binding protein that is essential for mtDNA maintenance, and analysis of mgm101 cells suggests that it is required for the repair of oxidative mtDNA damage (Chen et al., 1993; Meeusen et al., 1999). To gain insight into how nucleoids are organized and segregated within mitochondria in cells, we performed a cytological analysis of the behavior of nucleoid-associated components in vivo using fusions to fluorescent proteins.
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Results |
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To test whether the observed colocalization and interaction of Mmm1 and Mgm101 is functionally relevant for the maintenance of mtDNA in vivo, we examined whether mutant alleles of the genes encoding these proteins interact genetically. Specifically, we determined whether a combination of mmm1 (Burgess et al., 1994; mutation produces Mmm1G252A, see Materials and methods) and mgm101 (Meeusen et al., 1999; mutation produces Mgm101D131N) temperature-sensitive alleles in haploids produces a more severe defect in mtDNA maintenance than observed in haploid cells harboring only a single temperature-sensitive mutation, i.e., a synthetic defect. Maintenance of mtDNA was assessed by examining the growth of cells on the nonfermentable carbon source glycerol, where mtDNA is essential for viability, and by examining mtDNA-containing nucleoids in vivo directly by staining cells with DAPI. At permissive temperatures, mmm1ts and mgm101ts cells grew on glycerol and contained mtDNA nucleoids, indicating that the corresponding proteins were functional (Burgess et al., 1994; Meeusen et al., 1999; Table I). As expected, at nonpermissive temperature, none of the mutants were able to grow on glycerol, and mtDNA was undetectable by DAPI (Table I). Interestingly, haploid cells harboring a combination of mmm1ts and mgm101ts alleles displayed a significantly more severe mtDNA maintenance defect (Table I) as compared with mmm1ts or mgm101ts cells. Specifically, under permissive conditions, mmm1tsmgm101ts haploids were unable to grow on glycerol-containing media and were completely devoid of DAPI-stainable mtDNA nucleoid structures. The synthetic mtDNA stability defect observed in mmm1tsmgm101ts cells, coupled with our cytological and biochemical observations, is consistent with a model where outer membrane Mmm1 and matrix-localized Mgm101 function together within a TMS in the maintenance of mtDNA.
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To identify mtDNA replication foci, we used cells that contain an exogenous copy of a thymidine kinase gene and thus can phosphorylate and incorporate the thymidine analogue BrdU into their cellular DNA (Nunnari et al., 1997). Cells were pulse labeled for up to 30 min with BrdU, and mtDNA sites of incorporation were visualized by indirect immunofluorescence using a monoclonal anti-BrdU antibody (Nunnari et al., 1997). We have estimated by DAPI staining that a total of 42 ± 8 mtDNA-containing nucleoids are present per cell. In contrast, we detected by BrdU incorporation significantly fewer mtDNA replication foci per cell, indicating that mtDNA is replicating in only a subset of nucleoids at a given time (Fig. 4, A and B). Comparison of the number of mtDNA replication sites to the number of Mgm101 and Mmm1 foci in cells demonstrated a strong correlation, suggesting that TMSs are associated specifically with replicating mtDNA (Fig. 4 A). To directly determine the relationship of the TMS to mtDNA replication, we pulse labeled cells expressing Mgm101GFP or Mmm1GFP with BrdU. Strikingly, we observed that the vast majority mtDNA replication foci were colocalized with both Mgm101GFP and Mmm1GFP foci in cells (Fig. 4 B). These observations suggest that the autonomous TMS that we have identified functions as an mtDNA replisome.
One prediction of this model is that the mtDNA replication machinery is also a constituent of TMS. Thus, we determined the localization pattern of Mip1, the mtDNA polymerase. We observed that Mip1GFP, like Mgm101GFP, localized uniquely to a subset of DAPI-stained nucleoids (Fig. 4 A and not depicted) that coalign with Mmm1dsRED foci in the outer mitochondrial membrane in both rho+ and rhoo cells (Fig. 4 C and not depicted). These data indicate that the mtDNA polymerase also is a stable component of the nucleoid-associated TMS. Taken together, our findings suggest that mitochondria contain a two membranespanning autonomous structure that functions as a replisome.
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Discussion |
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It is interesting to speculate on what other functions TMS might perform in the cell. As proposed in the case of the B. subtilis replisome, TMS may function as a replication factory, harnessing the energy of nucleotide incorporation to drive the segregation/distribution of mitochondrial genomes throughout the organelle to ensure faithful DNA inheritance (Lemon and Grossman, 1998, 2000). In addition, the identification of Mgm101, a protein implicated in mtDNA repair (Meeusen et al., 1999), as a component of TMS raises the possibility that it may also function as an organizational center for mtDNA metabolic enzymes, not just those required for replication, thereby increasing the efficiency of events required for the maintenance of mtDNA.
The two membranespanning nature of the TMS suggests that it might serve to stably position the mtDNA maintenance machinery and mtDNA within the organelle through interactions with extramitochondrial components. Consistent with this notion, mutations in the outer membrane TMS component Mmm1 cause cortically localized mitochondrial tubules to collapse into centrally localized spherical structures (Burgess et al., 1994). This morphological phenotype associated with loss of Mmm1 function has been postulated to result from a loss of mitochondrial attachment to sites located at the cortex of the cell (Burgess et al., 1994). Interestingly, Mmm1 has also been shown to be required for actin binding to mitochondria in vitro, suggesting that Mmm1-dependent attachments may be to the actin cytoskeleton (Boldogh et al., 1998). However, we and others have observed that disassembly of the actin cytoskeleton using latrunculin-A does not affect the assembly or stability of TMS (Hobbs et al., 2001) or TMS movement in vivo (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200304040/DC1), suggesting that F-actin is not required for the putative TMS-dependent extramitochondrial attachment. Alternatively, the TMS may possess multiple unrelated functions within the cell: organization of mtDNA maintenance machinery and regulation of organelle structure.
Although the existence and nature of TMS extramitochondrial attachment sites are unknown, we have observed that TMSs have very limited movement within cells and that when movement is observed, in the majority of cases it is coupled to the movement of the mitochondrial organelle (90%, n = 30 nucleoids; Videos 2 and 3). An exception to this coordinated TMS/organelle movement is where the segregation of TMS within mitochondrial tubules was observed (Videos 2 and 3). Thus, our observations suggest that the organization of components responsible for maintenance of mtDNA into discrete structures whose behavior is membrane dependent may serve to secure the faithful inheritance of both the mtDNA metabolic machinery and associated mtDNA to daughter cells.
Our findings raise the question of whether TMS will be present in mitochondria of other cell types and function as a replisome and positioning apparatus. Recently, such a structure, termed tripartite attachment complex (TAC), was reported to exist in Trypanosoma brucei, where the mitochondrial genome is organized into a single copy kinetoplast that is attached to and segregated by the cell's basal body (Robinson and Gull, 1991; Ogbadoyi et al., 2003). Specifically, a differentiated region of mitochondrial outer and inner membrane between the kinetoplast and basal body was identified by EM analysis and shown to be a part of a superstructure of three distinct morphological regions, which include extra- and intramitochondrial filamentous structures that likely attach the kinetoplast to the basal body and function to position and segregate it (Ogbadoyi et al., 2003). The structural organization of TAC and TMS is similar, suggesting that like TAC, TMS functions to help position mtDNA in addition to its function as a replisome. Although trypanosomes are a specialized case where mtDNA is organized into a single copy structure that requires stringent segregation machinery, such as a basal body, our findings in yeast and other observations make it likely that mitochondrial TMSs also exist in mammalian cells. In human cells, similar nucleoid dynamics within the organelle have been recently reported using a GFP fusion to the helicase Twinkle (Garrido et al., 2003). In addition, in the absence of mtDNA, Twinkle retains its punctate morphology within mitochondrial tubules, suggesting that human mitochondria also contain mtDNA-independent structures dedicated to mtDNA maintenance (Spelbrink et al., 2001). Identification of additional TMS components and their organization within TMSs will lend insight into the nature of the link between mtDNA replication and inheritance and will ultimately enhance our understanding of mtDNA-linked human diseases.
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Materials and methods |
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Strains and plasmid construction
A previously characterized episomal mitochondrial-targeted MGM101GFP fusion (pTS330MGM101GFP) was used and transformed into the wild-type strain W303, yielding JNY 970 (Meeusen et al., 1999). We previously reported that 20-fold overexpression of Mgm101GFP caused the induction of respiratory-incompetent mitochondrial genomes (rho-) in wild-type cells (Meeusen et al., 1999). However, in this study, we expressed Mgm101GFP in wild-type cells at a level less than twofold above endogenous Mgm101p levels (not depicted). Under these experimental conditions, full respiratory competence and growth rates comparable to wild-type cells were observed (not depicted). An episomal mitochondrial-targeted ABF2GFP fusion (pts330ABF2GFP) was constructed by replacing MGM101 in pts330MGM101GFP with ABF2 by subcloning and transformed into W303 to yield JNY 969. Abf2GFP is fully functional, as assessed by its ability to complement the temperature-sensitive respiratory defect of abf2 cells (not depicted).
To generate an Mmm1dsRED fusion, pRS426MMM1GFP (provided by Steve Gorsich and Janet Shaw, University of Utah, Salt Lake City, UT) was digested with NheI and HpaI to drop out GFP, which was then replaced by dsRED.T1 (provided by Ben Glick, University of Chicago, Chicago, IL; Bevis and Glick, 2002) with engineered compatible ends: 5'-GCTAGCGCCTCCTCCGAGGACGTCATCAAGG-3' and 5'-TCCCCGGCTACAGGAACAGGTGGTGG-3'. Transformation of plasmids expressing Mmm1dsRED or Mmm1GFP into cells containing mmm1-1, a temperature-sensitive allele of MMM1 (provided by Rob Jensen, Johns Hopkins University, Baltimore, MD), resulted in full complementation of the glycerol growth defect at the nonpermissive temperature (Burgess et al., 1994, and not depicted). To generate MMM1:3XHA (JNY461), an integrating 3XHA cassette was PCR amplified with primers that were complimentary to the 3' region of MMM1 (Bahler et al., 1998). The PCR product was transformed into W303 cells, and transformants that integrated by homologous recombination at the MMM1 locus created an expressed in frame COOH-terminal Mmm1-3XHA. Correct integration was confirmed by PCR amplification across the recombination site. Mmm1-3XHA was fully functional, as assessed by the integrant's ability to grow on nonfermentable carbon sources at rates indistinguishable from wild-type strains (not depicted).
To generate MIP1:GFP (JNY967), an integrating GFP cassette was PCR amplified using primers that were complimentary to the 3' region of MIP1 (Bahler et al., 1998). The PCR product was transformed into W303 cells and integrated by homologous recombination at the MIP1 locus, creating an expressed in frame COOH-terminal Mip1GFP. Correct integration was confirmed by PCR amplification across the recombination site. Mip1GFP was fully functional, as assessed by the integrant's ability to grow on nonfermentable carbon sources at rates indistinguishable from wild-type stains (not depicted).
AFS98, a W303 strain harboring the thymidine kinase gene was constructed as reported by Nunnari et al. (1997). The plasmid containing ADH promoterregulated mitochondrial-targeted dsRed (PADHmitodsRED) was constructed as previously reported (Wong et al., 2000).
Haploid cells harboring combinations of the temperature-sensitive mgm101-2 (Meeusen et al., 1999) and mmm-1 (Burgess et al., 1994) alleles were obtained by crossing, sporulation, and tetrad analysis. As previously determined, mgm101-2 cells express a mutant Mgm101D131N protein. To determine the mutation in mmm-1 cells, we amplified the MMM1 locus in mutant cells by PCR using Vent polymerase (New England Biolabs, Inc.) and sequenced the products directly (Davis Sequencing, University of California, Davis). Sequencing revealed a single point mutation at the MMM1 locus from G to A, resulting in a change in amino acid 252 from G to S. Characterization of the glycerol growth defects on solid YPD and YPG media and determination of DAPI-stainable nucleoids in mutants were performed as described by Meeusen et al. (1999).
Immunoprecipitation of cross-linked mitochondrial proteins
To enrich for mitochondrial proteins, mitochondria were isolated from W303 and JNY461 by differential centrifugation as previously described (Meeusen et al., 1999). Cross-linking of mitochondrial proteins and immunoprecipitations were conducted as described by Wong et al. (2003). Anti-HA antibodies were purchased from Covance Inc., anti-Mgm101 antibodies were prepared as previously described (Meeusen et al., 1999), and anti-Tim23 antibodies were a gift from Rob Jensen.
BrdU incorporation and detection by indirect immunofluorescence
AFS98 cells harboring pts330MGM101GFP were cultured to OD600 0.2 in YPG overnight and shifted to YPDGal + 1 µg/ml DAPI for 20' at 25°C to induce Mgm101GFP expression. Cells were then immediately washed into YPD containing 5 mg/ml sulfanilamide (Sigma-Aldrich), 100 µg/ml amethopterin (Sigma-Aldrich, from a 100x stock in dimethyl sulfoxide), and 500 µg/ml BrdU for 30 min at 30°C. Cells were then washed three times in YPD and fixed by resuspension in YPD + 3.7% formaldehyde for 2 h at 25°C.
Cells were then processed as previously described (Nunnari et al., 1997) with the following modifications to preserve GFP signal. After adhering cells to slides, cells were incubated with PBS containing 0.5% Tween 20 (Sigma-Aldrich) for 30 min, 0.3 N HCl for 5 min, 0.1 M sodium tetraborate, pH 8.5, for 5 min, before washing one time in PBS. Indirect immunofluorescence was conducted as previously described, substituting the fluorescein-conjugated antimouse secondary antibody with a rhodamine-conjugated antimouse secondary antibody (Molecular Probes).
Fluorescence microscopy imaging
Yeast strains were grown either in YPG or YPD, in the case of rhoO cells, overnight to early log phase, pelleted, and resuspended in YPDGal in either the presence or absence of 1 µg/ml DAPI for 2060 min at 25°C before visualization using fluorescence microscopy. To visualize the mitochondrial organelle, strains were transformed with pADHmitodsRED or stained with Mitotracker CMXR (Molecular Probes) as previously described (Nunnari et al., 1997).
Cells were viewed with an Olympus IX70 Deltavision Microscope using a 60x 1.4 N.A. objective and a 100-W mercury lamp. The following excitation wavelengths were used: DAPI, 360; FITC, 490; and Rhod, 555. Images were collected in 0.2-µm sections. Two- and three-dimensional light microscopy data collection and computational removal of out-of-focus information used an integrated, cooled CCD-based, fluorescence light microscopy data collection, processing, and visualization workstation (Applied Precision, Inc.) in the Molecular and Cellular Biology Imaging Facility, University of California, Davis. Three-dimensional datasets were processed using DeltaVision's iterative, constrained three-dimensional deconvolution method. Time-lapse analyses were done with a Princeton Micromax Camera equipped with a Sony Interline Chip. As part of our analysis of time-lapse data for Videos 2 and 3, we examined complete z-section series for every time point to rule out the possibility that movement in the z-axis of another unrelated TMS gave rise to two TMS foci as opposed to segregation of a single TMS.
Online supplemental material
The supplemental material (Videos 14) is available at http://www.jcb.org/cgi/content/full/jcb.200304040/DC1. Supplementary videos show time-lapse microscopy of the behavior of TMS components and mitochondria in live cells either containing or lacking mtDNA or treated with latrunculin.
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Acknowledgments |
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J. Nunnari is supported by grants from the National Institutes of Health (NIH) (R01GM62942A) and the National Science Foundation. S. Meeusen has received support from an NIH training grant (G.M.-07377).
Submitted: 7 April 2003
Accepted: 16 September 2003
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