1 Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
2 Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan
*Author for correspondence (e-mail: endo{at}biochem.chem.nagoya-u.ac.jp)
Accepted June 23, 2001
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SUMMARY |
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Key words: Mitochondrial morphology, Hsp70, Yeast
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INTRODUCTION |
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Mitochondria share common double-membrane structures characterized by specific distribution of mitochondrial proteins, but their morphology and copy numbers change significantly and frequently (e.g. in response to growth conditions and during cellular differentiation and development) (Pon and Schatz, 1991; Hermann and Shaw, 1998; Yaffe, 1999). These morphological alterations are often associated with branching, stretching, shrinking, fission and fusion of tubular structures.
Yeast provides a useful model system to study dynamics of mitochondrial morphology. Recent yeast genetic screen analyses led to identification of several proteins involved in the maintenance of mitochondrial morphology and mitochondrial inheritance during mitotic division. Identification of mitochondrial outer membrane proteins (Burgess et al., 1994; Sogo and Yaffe, 1994; Berger et al., 1997; Boldogh et al., 1998; Shepard and Yaffe, 1999) and cytoskeletal elements including actin and intermediate filaments (Drubin et al., 1993; Hermann et al., 1997; McConnell and Yaffe, 1992) as components responsible for mitochondrial distribution and inheritance in yeast cells led to the proposal that interactions between the mitochondrial outer membrane and cytoskeleton are important for the mitochondrial morphology and inheritance. In addition, it is evident that a balance between division and fusion of mitochondria maintains the normal mitochondrial morphology. Fzo1p and Dnm1p, two GTP-binding proteins on the mitochondrial surface, regulate fusion and fission of mitochondrial tubules, respectively (Hermann et al., 1998; Otsuga et al., 1998; Bleazard et al., 1999; Sesaki and Jensen, 1999), and protiens that regulate the mitochondrial division in cooperation with Dnm1p have been identified (Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001). By contrast, little is known about possible involvement of mitochondrial matrix proteins in the control of mitochondrial morphology. In connection to this, it was observed that depletion of Yge1p leads to aggregation of mitochondria in yeast cells, suggesting the role of the Hsp70 chaperone system in the maintenance of mitochondrial morphology (Ikeda et al., 1994).
In this study, we analyzed the effects of loss of mtHsp70 functions on mitochondrial morphology by using mtHsp70 temperature-sensitive mutant and mdj1 mutant strains. We observed that defects in the functions of mtHsp70 led to aggregation of mitochondria in cells. This mitochondrial aggregation was not due to indirect effects caused by defects in protein import into mitochondria. Instead, the results suggest that the mitochondrial Hsp70 system is essential for optimizing the functions of as-yet-unidentified heat-labile protein in the mitochondrial matrix in controlling the mitochondrial morphology.
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MATERIALS AND METHODS |
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Plasmids and strain constructions
Standard recombinant techniques were used with Escherichia coli strain TG1 (supE hsd5 thi
(lac-proAB) F'[traD36 proAB+ lacIq lacZ
M15]. The S65T-GFP gene (Heim et al., 1995) was amplified by PCR using primers 5'-GCGCTCGAGAATGGGTAAAGGAGAAGAA-3' and 5'-CGCAAGCTTATCTAGATCCGGACTTG-3'. The amplified 0.7-kb fragment was digested with XhoI and HindIII and introduced into the XhoI and HindIII sites of pCI-X22D/SP, which contains the pCOXIV-DHFR fusion gene (Hurt et al., 1984). The resulting plasmid, pAK1, was digested with EcoRI and HindIII and the 0.8 kb fragment containing the pCOXIV-S65TGFP fusion gene was introduced into the EcoRI and HindIII sites of YCpUG578T (a single copy plasmid containing the GAL1 promoter and the URA3 gene; Y. Ohya, personal communication) to produce pAK2.
The MDJ1 gene was cloned by PCR from yeast genomic DNA using primers 5'-TACTCTCCGTCTAGAGTG-3' and 5'-CTATTCTCTCGAGCTCAG-3'. The amplified 2.5-kb DNA fragment was digested with XhoI and XbaI, and introduced into the XhoI and the XbaI sites of pBluescript II SK+ (Stratagene) to give pAK23. A 0.8 kb SmaI/StuI fragment of pJJ281 (Jones and Prakash, 1990) containing the TRP1 gene was introduced into the EcoRV sites of pAK23 to generate pAK24-1. A 2.1 kb XhoI/HincII fragment of pAK24-1 was used to transform the yeast strain, SEY6210. Trp+ transformants were selected, and the presence of the mdj1 allele was confirmed by PCR and immunoblotting using anti-Mdj1p antibodies. The resulting
mdj1 strain was named AKSC7-1. To construct the GAL1-MDJ1 hybrid gene, a 1.5 kb DNA fragment was amplified by PCR using primers 5'-GCGAGATCTATGGCTTTCCAACAAGGTG-3' and 5'-GCGAAGCTTAATTTTTTTTGTGTCACCTTTG-3'. The amplified DNA fragment was digested with BglII and HindIII and introduced into the BglII and the HindIII sites of YCpUG578T to give pAK27.
Fluorescence microscopy
Immunofluorescent staining of yeast cells was performed as described previously (Nishikawa et al., 1994). The rabbit anti-Tom40 antiserum was used as a primary antibody at 1:500 dilution and fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG (ICN Pharmaceuticals, Aurora, OH) as a secondary antibody. For monitoring GFP fluorescence, cells were fixed in the same manner as described previously (Nishikawa et al., 1994). Rhodamine-phalloidin staining of actin in yeast cells was performed as described (Sekiya-Kawasaki et al., 1998). Cells were viewed on an Olympus BH-2 epifluorescent microscope (Olympus, Tokyo) with a filter set suitable for fluorescein and photographed with T-MAX 400 film (Eastman Kodak, Rochester, NY) developed at ASA1600. Cells were also observed using an inverted fluorescence microscope (IX70; Olympus) equipped with a fluorescein filter set. In this case, cell images were taken by using a cooled CCD camera system (MicroMAX; Princeton Research Instruments) with IPLab image processing software (Scanalytics).
Electron microscopy
Yeast cells were grown to an early log phase in YPD medium at 23°C. The cultures were transferred to 37°C and further incubated for 60 minutes with aeration. Permanganate fixation was performed as described (Kaiser and Schekman, 1990) except that the uranyl acetate staining step was omitted. Cells were embedded in Spurrs resin and sections were cut to 60 nm, which were subsequently stained with 3% uranyl acetate for 2 hours and Reynolds lead citrate for 10 minutes. The sections were examined and photographed on a JEOL2010 transmission electron microscope at 100 kV.
Immunoblotting
Crude yeast cell extracts were prepared by trichloroacetic acid precipitation method (Yaffe and Schatz, 1984). Immunoblotting was performed using Cy5-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech) as a secondary antibody and analyzed with Storm 860 image analyzer (Molecular Dynamics).
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RESULTS |
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Is the aberrant mitochondrial morphology observed in the ssc1-2 and ssc1-3 mutant cells distinct from the collapsed mitochondria observed previously for the mmm1 mutant at 37°C (Burgess et al., 1994)? Immunofluorescent staining of mmm1 mutant cells with anti-Tom40 antibodies showed bright staining of the mitochondrial rim at 37°C (Fig. 2d), indicating that mitochondria are converted to giant spherical mitochondria, as reported previously (Burgess et al., 1994). This staining is clearly different from that observed in the mtHsp70 mutants and suggests that inactivation of the mtHsp70 activity does not convert mitochondrial networks to a spherical shape. Immunofluorescent staining of dnm1 mutant cells showed networks of interconnected mitochondria (not shown) (Sesaki and Jensen, 1999), which are different from the aggregated mitochondrial structure observed for the ssc1 mutant cells.
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Next, we asked if functional defects of the mitochondrial protein translocators themselves caused aggregation of mitochondria. The inner membrane proteins, Tim23 and Tim17, are the essential subunits of TIM, the translocase of the mitochondrial inner membrane (Emtage and Jensen, 1993; Dekker et al., 1993; Kübrich et al., 1994; Ryan et al., 1994). Ssc1p mediates unidirectional protein translocation through the channel of the Tim17/23 complex into the matrix (Kronidou et al., 1994; Schneider et al., 1994; Berthold et al., 1995). We previously isolated a temperature-sensitive mutant allele of the TIM23 gene that is defective in protein translocation across the inner membrane at restrictive temperature (S.N. and T.E., unpublished). Mitochondrial structures in the tim23 mutant and the isogenic wild-type cells were visualized by expressing a fusion protein between the mitochondrial targeting presequence and green florescent protein (pCOXIV-S65TGFP). Normal tubular networks of mitochondria were observed in both tim23 mutant cells and wild-type cells at 23°C (Fig. 5Aa,c). When incubated at 37°C for 2 hours, pF1ß was accumulated in tim23 mutant cells (not shown), indicating the blockage of protein translocation across the inner membrane. However, normal mitochondrial tubular networks still remained around the cellular periphery in the tim23 mutant cells after incubation at 37°C for 2 hours (Fig. 5Ad). Essentially the same results were obtained when mitochondria were stained with anti-F1ß antibodies (not shown). These results indicate that defects in the functions of mitochondrial translocators in the mitochondrial membranes themselves do not cause mitochondrial aggregation.
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It was reported that deletion of the MDJ1 gene causes loss of mitochondrial DNA (Rowley et al., 1994), so that the cells become [rho0]. However, as reported by Guan et al., the loss of mitochondrial DNA itself does not affect the normal tubular mitochondrial structures (Guan et al., 1993); we observed extended tubular mitochondrial structures around the cell periphery in [rho0] cells isogenic to the mdj1 mutant (Fig. 5Bb). Further, the mitochondrial aggregation in the
mdj1 mutant cells was completely rescued when the MDJ1 gene was placed under the GAL1 promoter and Mdj1p was expressed (data not shown). Therefore, it is unlikely that the mitochondrial aggregation in the
mdj1 mutant merely reflects the secondary effects arising from the loss of mitochondrial DNA.
The results obtained here suggest that mitochondrial aggregation observed in the mdj1 mutant and in the mtHsp70 mutants is most likely due to the defects in prevention of protein aggregation in the mitochondrial matrix. If the presence of irreversible aggregates or an inclusion body consisting of heat-labile matrix proteins nonspecifically causes aberrant mitochondrial morphology, the morphological defects will not be recovered after temperature downshift from 37°C to 23°C. However, this is not the case because aggregation of mitochondria in
mdj1 cells after incubation at 37°C was relieved when temperature was shifted down to 23°C. The fraction of cells containing aggregated mitochondria decreased to 35% within 2 hours after temperature downshift (Fig. 6D). This relatively rapid recovery of mitochondrial morphology suggests that aggregated mitochondria still have an ability to regain their normal extended mitochondrial networks. When cycloheximide was added to the medium upon temperature downshift, recovery of the normal mitochondrial morphology was inhibited (Fig. 6C), suggesting that the recovery process requires de novo protein synthesis.
Depolymerization of actin cables partially suppresses mitochondrial aggregation induced by inactivation of the mtHsp70 activity
Depolymerization of the actin cytoskelton alters normal mitochondrial morphology (Boldogh et al., 1998). To rule out the possibility that mitochondrial aggregation in the mutants of the mtHsp70 system was due to the secondary effects of the disturbed actin cytoskeleton, we examined the morphology of the actin cytoskeleton in the ssc1 and mdj1 mutants. Distribution of the actin patches and cables in mtHsp70 and
mdj1 mutant cells are similar to that in wild-type cells both at 23°C and at 37°C (Fig. 7). This means that loss of the mtHsp70 activity did not cause a significant morphological change of the actin cytoskeleton.
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DISCUSSION |
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The mitochondrial aggregation caused by the defective mtHsp70 system does not arise from impaired interactions of mitochondria with the actin cytoskeleton and is not the consequence of the block of protein translocation across the mitochondrial membranes. Instead, mitochondrial aggregation in mtHsp70 cells may be induced by defects in the ability of mtHsp70 to prevent irreversible protein inactivation during folding/assembly at low temperatures or denaturation at elevated temperatures. In other words, the results suggest that the mitochondrial matrix contains at least one protein that is responsible for normal mitochondrial morphology and requires the mitochondrial Hsp70 system for its function in a wide range of temperatures, although such a matrix protein has not yet been identified. This matrix protein appears to be heat labile because mitochondrial aggregation is enhanced at elevated temperature.
How can a protein in the mitochondrial matrix control mitochondrial morphology? One possibility is that the matrix protein may form a fibrous structure in the mitochondrial interior that serves to stabilize the elongated shape of mitochondria. FtsZ forms a cytoskeletal framework of the cytokinetic ring in bacterial cells, which functions as an essential component for the bacterial cell division (Erickson, 1997). Chloroplasts also use FtsZ homologs for their division (Osteryoung et al., 1998; Strepp et al., 1998). However, no gene for a FtsZ homolog is found in the yeast genome. Nevertheless electron microscopy revealed the presence of an intramitochondrial fibrous component in various yeast species (Yotsuyanagi, 1988). This filament-like structure was observed as multiple layers that span the longitudinal axis of a mitochondrial body. Although a protein that assembles to form the intramitocondrial fibrous structure has not yet been identified, mtHsp70 probably assists its initial folding after being imported into the matrix and prevents it from heat damage at elevated temperatures. Further, mtHsp70 may participate directly in the assembly and/or disassembly of the fibrous structure at physiological temperature (e.g. 23°C). Such a role may be compared with that of cytosolic Hsp70 in microtuble formation/dissociation (Oka et al., 1998). Our laboratory is currently searching for a mitochondrial matrix protein that is involved in the maintenance of normal mitochondrial morphology.
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ACKNOWLEDGMENTS |
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