Correspondence to David C. Chan: dchan{at}caltech.edu
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Introduction |
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The assembly of functional Dnm1p complexes on mitochondria is a critical issue in understanding the mechanism of mitochondrial fission. The mitochondrial outer membrane protein Fis1p is required for the formation of normal Dnm1p puncta on mitochondria. In fis1 cells, Dnm1p puncta are primarily cytosolic or form abnormally large aggregates on mitochondria (Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000). Mdv1p interacts with Fis1p through its NH2-terminal half and with Dnm1p through its COOH-terminal WD40 domain. However, Mdv1p appears dispensable for Dnm1p assembly on mitochondria because mdv1
cells show little or no change in Dnm1p localization, even though mitochondrial fission is disrupted (Fekkes et al., 2000; Tieu and Nunnari, 2000; Tieu et al., 2002; Cerveny and Jensen, 2003). These observations have led to two important features of a recently proposed model for mitochondrial fission (Shaw and Nunnari, 2002; Tieu et al., 2002; Osteryoung and Nunnari, 2003). First, Fis1p acts to assemble and distribute Dnm1p on mitochondria in an Mdv1p-independent step. Second, Mdv1p acts downstream of Dnm1p localization to stimulate membrane scission. An alternative model proposes that Dnm1p marks the site of mitochondrial fission and recruits Fis1p and Mdv1p into an active fission complex (Cerveny and Jensen, 2003). Again, in this model Mdv1p functions downstream of Dnm1p localization.
Despite extensive efforts, however, there is no evidence that Fis1p can interact directly with Dnm1p. We speculated that there may be an additional component of the mitochondrial fission pathway required for the Fis1p-dependent assembly of Dnm1p puncta on mitochondria. Because a genome-wide screen for mitochondrial morphology mutants (Dimmer et al., 2002) did not yield obvious candidates, we used a biochemical approach to identify additional components of the mitochondrial fission machinery. Using immunopurification and mass spectrometry, we have identified the WD40 repeat protein Caf4p as a Fis1p-interacting protein. Caf4p localizes to mitochondria and associates with Fis1p, Mdv1p, and Dnm1p. Moreover, we show that mdv1 cells are only partially deficient in mitochondrial fission due to the redundant activity of Caf4p. Importantly, Caf4p mediates recruitment of Dnm1p puncta to mitochondria in mdv1
yeast. Inclusion of CAF4 significantly clarifies the current models for mitochondrial fission.
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Results |
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Interestingly, peptides derived from the WD40 repeat protein Caf4p were identified in both Fis1p MudPIT experiments (24.4% coverage, 9 unique peptides; 8.5% coverage, 3 unique peptides). CAF4 (YKR036C) was first identified in a yeast two-hybrid screen for CCR4p-interacting proteins (Liu et al., 2001). CCR4p is a central component of the CCR4-NOT transcriptional regulator and cytosolic deadenylase complex (Denis and Chen, 2003). Caf4p is the nearest homologue of Mdv1p in S. cerevisiae (38% identity and 57% similarity), and the two proteins show extensive sequence identity throughout their lengths (Fig. 1 B). Both proteins share a unique NH2-terminal extension (NTE) (25.3% identity), a central coiled-coil (CC) domain (19% identity) and a COOH-terminal WD40 repeat domain (44.4% identity). The Caf4p CC scores significantly more weakly (0.3 probability) than the Mdv1p coiled coil (
1.0 probability) in the MultiCoil prediction program (Wolf et al., 1997).
Caf4p interacts with components of the mitochondrial fission machinery
We sought independent confirmation of the physical interaction between Fis1p and Caf4p. For immunoprecipitation experiments, Caf4p-HA or Mdv1p-HA were expressed from their endogenous promoters in strains carrying chromosomal M3TH-FIS1 (3XMyc/TEV/His8-FIS1) and deleted for CAF4 or MDV1, respectively. When M3TH-Fis1p was immunoprecipitated, 5% of both Caf4p-HA and Mdv1p-HA coprecipitated (Fig. 2 A, lanes 7 and 10).
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We also used a yeast two-hybrid assay to analyze the interaction of Caf4p and Mdv1p with Fis1p and Dnm1p (Table I). Full-length Caf4p and an NTE/CC fragment of Caf4p interacted strongly with the cytosolic portion of Fis1p (residues 1128), consistent with our immunoprecipitation data. Similar interactions were observed between Fis1p and both full-length Mdv1p and the NTE/CC region of Mdv1p, as has been previously reported (Tieu et al., 2002; Cerveny and Jensen, 2003). The WD40 domain of both Mdv1p and Caf4p interacted strongly with Dnm1p. However, full-length Mdv1p interacted more weakly and an interaction between full-length Caf4p and Dnm1p was not detected. These results suggest that the interaction of the WD40 domain with Dnm1p is regulated and may be inhibited by the NH2-terminal region of Caf4p and Mdv1p.
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Caf4p is involved in mitochondrial division
Given that Caf4p interacts with Fis1p, Mdv1p, and Dnm1p, we hypothesized that Caf4p, like Mdv1p, is a component of the mitochondrial division apparatus. caf4 yeast, however, display normal mitochondrial morphology, with tubular mitochondria evenly dispersed around the cell cortex (Fig. 3). Wild-type mitochondrial morphology was also observed at elevated temperatures and on carbon sources other than dextrose (glycerol or galactose; unpublished data). This observation is not surprising, given that CAF4 was not identified in a genome-wide screen of deletion strains for mitochondrial morphology mutants (Dimmer et al., 2002).
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Most interestingly, we found that mdv1 caf4
cells have mitochondrial net distributions indistinguishable from either dnm1
cells or fis1
cells. Deletion of CAF4 in mdv1
cells markedly shifts the distribution to one composed almost entirely of collapsed mitochondrial nets (>90% in dextrose, Fig. 3). Our results support a model in which partial reduction of mitochondrial fission results in predominantly spread mitochondrial nets, and complete loss of fission eventually results in collapse of the nets. That is, mdv1
cells retain residual mitochondrial fission, whereas mdv1
caf4
cells are devoid of fission, similar to dnm1
, fis1
, or mdv1
dnm1
cells. An analogous situation appears to exist in mammalian cells, in which weak Drp1 dominant-negative alleles cause the formation of spread nets, whereas strong dominant-negative alleles cause nets to collapse (Smirnova et al., 2001).
We tested this model by reanalyzing mitochondrial morphologies in the presence of latrunculin A, which disrupts the actin cytoskeleton. Disruption of the actin cytoskeleton leads to rapid fragmentation of the mitochondrial network due to ongoing mitochondrial fission (Boldogh et al., 1998; Jensen et al., 2000). Latrunculin A treatment rapidly resolves a fraction of collapsed nets into spread nets (Jensen et al., 2000; Cerveny et al., 2001), and allows a closer examination of the degree of connectivity in mitochondrial nets. Similarly, in mammalian cells, collapsed mitochondrial nets induced by overexpression of dominant-negative Drp1 can be spread by the microtubule-depolymerizing agent nocodazole (Smirnova et al., 2001). Both wild-type and caf4 yeast treated with latrunculin A show mitochondrial fragmentation (Fig. 4). 80% of mdv1
cells treated with latrunculin A contain partial mitochondrial nets (Fig. 4 E, partial net) that are less interconnected and have fewer fenestrations than the collapsed or spread nets that predominate in latrunculin Atreated dnm1
or fis1
cells. 95% of latrunculin Atreated mdv1
caf4
cells show either collapsed nets or highly fenestrated spread nets, a profile indistinguishable from that in dnm1
or fis1
cells (Fig. 4). Thus, after disruption of the actin cytoskeleton, mdv1
yeast display a distribution of mitochondrial morphologies that suggest an incomplete defect in mitochondrial fission. In contrast, mdv1
caf4
yeast have mitochondrial morphologies similar to that in fis1
and dnm1
yeast. We conclude that CAF4 mediates low levels of mitochondrial fission in mdv1
cells.
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Mitochondrial fission is blocked by overexpression of Caf4p or Caf4p fragments
Because overexpression of Mdv1p or Mdv1p fragments inhibits mitochondrial fission (Cerveny and Jensen, 2003), we next tested the effects of Caf4p overproduction. Caf4p-HA under the control of the GalL promoter was expressed 20 times above endogenous levels in rich galactose medium (unpublished data). Spread mitochondrial nets formed in 23.5% of cells (Fig. 5 C). An additional 38% of cells had an intermediate phenotype that we termed "connected tubules," consisting of a completely interconnected mitochondrial network in which no tubular ends were detected (Fig. 5 B). Overexpression of an NH2-terminal fragment that interacts with Fis1p (residues 1250; unpublished data) had a similar effect (9% spread nets, 33% connected tubules; Fig. 5), suggesting that the formation of mitochondrial net-like structures may result from a dominant-negative effect on Fis1p function. A similar distribution of mitochondrial phenotypes resulted from 20-fold overproduction of Mdv1p-HA (7.5% spread nets and 24.5% interconnected tubules) and an Mdv1p-HA NH2-terminal fragment (5% spread nets and 39% interconnected tubules; unpublished data). These data confirm that Caf4p interacts with the mitochondrial fission apparatus.
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Caf4p recruits Dnm1p-GFP to mitochondria
To understand the mechanism of mitochondrial fission, it is crucial to elucidate how Dnm1p is recruited to mitochondria. Given that Mdv1p associates with both Fis1p and Dnm1p, it is puzzling that Dnm1p assembly on mitochondria shows little or no dependence on Mdv1p (Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000; Tieu et al., 2002; Cerveny and Jensen, 2003). With the identification of Caf4p as a component of the fission machinery, we reexamined this issue. We constructed a fully functional Dnm1p-GFP allele and analyzed its localization pattern using deconvolution microscopy (Table II). Similar to previous reports (Otsuga et al., 1998), Dnm1p-GFP is found predominantly in puncta associated with mitochondria (average 16.9 mitochondrial vs. 3.3 cytosolic puncta per cell) (Table II and Fig. 8, AC). Deletion of CAF4 or MDV1 alone had little effect on this localization (15.4 mitochondrial vs. 5.2 cytosolic and 13.7 mitochondrial vs. 5.1 cytosolic per cell, respectively; Table II and Fig. 8, DI). In all these strains, the Dnm1p puncta are relatively uniform in size and intensity.
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Discussion |
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Genetically, both MDV1 and CAF4 act positively in the mitochondrial fission pathway. mdv1 cells are dramatically compromised for mitochondrial fission, but a residual level of fission is mediated by CAF4. This residual fission activity is revealed by the observation that mdv1
yeast have a less severe mitochondrial morphology defect compared with fis1
or dnm1
yeast. In contrast, mdv1
caf4
yeast display predominantly collapsed mitochondrial nets, identical to those seen in fis1
and dnm1
cells. Time-lapse imaging of mitochondria in mdv1
cells indeed reveals a residual level of fission that is absent from mdv1
caf4
cells. These results directly support our conclusion that the morphology differences between mdv1
cells versus mdv1
caf4
, fis1
, and dnm1
cells are primarily due to differences in fission rates. It is also possible that the proposed role of Dnm1p in cortical distribution of mitochondria may contribute in part to the morphological differences (Otsuga et al., 1998). The mdv1
mutation acts as a weak suppressor of the glycerol growth defect in fzo1
cells. The mdv1
caf4
double mutation suppresses this phenotype much more efficiently. Based on these physical interaction and genetic data, we conclude that Caf4p likely acts in a similar manner to Mdv1p to promote mitochondrial fission.
Why are there two proteins that appear to perform similar and partially redundant roles in mitochondrial fission? This question is particularly intriguing because caf4 yeast have normal mitochondrial morphology, indicating that disruption of Caf4p does not cause a major loss of mitochondrial fission. First, CAF4 may play a more important role in mitochondrial fission under conditions not yet tested. Second, the presence of two proteins mediating interactions between Fis1p and Dnm1p would increase the ability of cells to accurately regulate the rate of mitochondrial fission. The heterotypic and homotypic interactions between Caf4p and Mdv1p may provide an additional layer of regulation. Finally, Caf4p may have an additional function in another pathway. Previous two-hybrid studies have implicated Caf4p in the CCR4-NOT complex, which is thought to be involved in regulation of transcription and/or mRNA processing (Liu et al., 2001).
A revised model for mitochondrial fission
The current models for mitochondrial fission propose that Mdv1p acts late in the fission pathway. One model proposes a two-step pathway in which Fis1p first recruits Dnm1p, in an Mdv1p-independent manner. Mdv1p then acts as a molecular adaptor at a post-recruitment step, along with Fis1p, to promote fission by Dnm1p (Shaw and Nunnari, 2002; Tieu et al., 2002; Osteryoung and Nunnari, 2003). A second model also proposes that Mdv1p acts after Dnm1p recruitment to organize an active fission complex (Cerveny and Jensen, 2003).
Our study reveals a new role for Mdv1p and Caf4p early in mitochondrial fission. Fis1p recruits Dnm1p to mitochondrial fission complexes through Mdv1p or Caf4p, which act as molecular adaptors. This revised model is strongly supported by our demonstration that Dnm1p recruitment in mdv1 yeast depends on Caf4p function. In the absence of both Mdv1p and Caf4p, Fis1p is unable to recruit Dnm1p.
Although Mdv1p and Caf4p clearly act early in the fission pathway, there is evidence that at least Mdv1p has a subsequent role in the activation of fission, as previously proposed (Shaw and Nunnari, 2002; Tieu et al., 2002; Cerveny and Jensen, 2003). In caf4 cells, Mdv1p recruits Dnm1p to fission complexes, and fission occurs at apparently normal levels. However, in mdv1
cells, Caf4p is similarly able to recruit Dnm1p to fission complexes, but mitochondrial fission is severely compromised. Therefore, Mdv1p and Caf4p can independently recruit Dnm1p, but complexes recruited by Mdv1p appear to be more highly active. These observations suggest that Dnm1p recruitment by itself is insufficient for fission to occur. Indeed, studies of Dnm1p dynamics indicates that most Dnm1p puncta do not result in fission (Legesse-Miller et al., 2003). Our identification of Caf4p as part of the fission machinery clarifies the early steps in mitochondrial fission. Future studies will need to define the additional steps beyond Dnm1p recruitment necessary for fission.
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Materials and methods |
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Plasmid construction
The M9TH cassette was generated as follows. Primers Eg258 (see Table S3, available at http://www.jcb.org/cgi/content/full/jcb.200503148/DC1) and Eg259 were used to amplify URA3 from pRS416 (Stratagene). Eg260 and Eg4, an FZO1 reverse primer, were used to amplify a TEV/His8 module from EG704 (pRS414 + 9XMyc/TEV/His8-FZO1). The 3' end of the URA3 product overlaps by 18 bp with the 5' end of the TEV/His8 product. This overlap allows them to anneal together and be amplified in a second PCR with the primers Eg258 and Eg4. The URA3/TEV/His8 product was cloned into pRS403 as an EcoRV/SalI fragment (which removes all FZO1 sequence), resulting in EG928. 9xMyc/TEV was amplified with Eg256 and Eg260 from EG704 and fused to the 5' end of URA3 (Eg258/259 product) by mixing and amplifying with Eg256 and Eg259. The resulting product was cloned into EG928 as an EcoRV/EcoRI fragment, yielding EG940 (pRS403+9xMyc/TEV/URA3/TEV/His8). EG940 was converted to pRS403+3xMyc/TEV/URA3/TEV/His8 by digesting with Xba1, yielding EG957.
To construct HA-tagged versions of CAF4 and CAF4 fragments, CAF4 sequences were PCR amplified from end3 genomic DNA (Open Biosystems). First, the CAF4 3' untranslated region (UTR) was amplified with the primers Eg313 and Eg314 and cloned as a KpnI/SalI fragment into pRS416, resulting in pRS416 + CAF4 3' UTR. 3XHA was amplified with Eg327 and Eg328 and cloned as a SalI/XhoI fragment into the SalI site to generate pRS416 + 3XHA/CAF4 3' UTR. The CAF4 5' UTR was cloned as a SacI/SpeI fragment using Eg312 and Eg317, resulting in pRS416 + CAF4 5' UTR/3XHA/3' UTR. Full-length CAF4 was amplified with Eg316 and Eg315 and cloned as a SpeI/XhoI fragment into the SpeI/SalI sites, resulting in EG1041. CAF4 N (residues 1274) and C (residues 275659) were amplified with Eg316/Eg353 and Eg315/Eg352, respectively, and cloned as SpeI/XhoI fragments, resulting in EG1045 and EG1043. Four independent clones encoded glutamine at residue 110 and arginine at residue 111. Full-length CAF4-HA was able to complement caf4
in caf4
mdv1
yeast, indicating that it is functional.
To construct HA-tagged versions of MDV1 and MDV1 fragments, MDV1 sequences were amplified by PCR from end3 genomic DNA. First, the MDV1 3' UTR was amplified with the primers Eg323 and Eg324 and cloned as a SacI/SalI fragment into pRS416. A 3XHA cassette was added as described for CAF4-HA, resulting in the plasmid pRS416 + 3XHA-MDV1 3' UTR. The MDV1 5' UTR was amplified with primers Eg320 and Eg322 and cloned as a SacII/SpeI fragment, resulting in pRS416 + MDV1 5' UTR/3XHA/3' UTR. Full-length MDV1 was amplified using primers Eg109 and Eg321 and cloned as a SpeI/XhoI fragment into the SpeI/SalI sites, resulting in EG1047. MDV1 N (residues 1300) and C (residues 301714) were amplified with Eg323/Eg326 and Eg321/325, respectively, and cloned as SpeI/XhoI fragments, resulting in EG1051 and EG1049. Full-length MDV1-HA complemented the mitochondrial morphology defects in mdv1
cells.
The galactose-inducible Caf4p expression vectors EG1133 (Caf4p-HA), EG1135 (Caf4p-HA, residues 251659), and EG1136 (Caf4p-HA, residues 1250) were generated by replacing the CAF4 5' UTR in EG1041, EG1043, and EG1045 with a SacI/ClaI GalL promoter fragment from p413 GalL (Mumberg et al., 1994) containing a start codon inserted between the XbaI and EcoRI sites.
pRS403 + GPD/mito-GFP (EG686) was generated by first cloning the GPD promoter from p413 GPD (Mumberg et al., 1995) as a SacI (blunt)/SpeI fragment into the SmaI/SpeI sites of pRS403 (Stratagene), yielding EG128. Next, a HindIII (blunt)/NotI mito-GFP fragment from pYES-mtGFP (Westermann and Neupert, 2000) was inserted into EG128 linearized with SpeI (blunt)/NotI. pRS403 + GPD/mito-DsRed (EG823) was generated by subcloning DsRed into the BamHI and NotI sites of EG686, replacing GFP with DsRed. OM45 was PCR amplified with primers Eg151 and Eg154 and cloned as an XhoI/XbaI fragment with an XbaI/BamHI GFP fragment into the XhoI/BamHI sites of pRS416, yielding pRS416 + OM45-GFP (EG252).
Yeast strain construction
An M9TH-FIS1 strain was generated by amplifying the 9XMyc/TEV/URA3/TEV/His8 cassette from EG943 (pRS403-9XMyc/TEV/URA3/TEV/His8) with the FIS1-targeting primers Eg261 and Eg262 and transforming YJG12. URA3+ transformants were screened by PCR for correct integration (2 out of 8 positive), grown overnight in YPD to allow for loss of URA3, and plated on 5-FOA plates. Colonies were screened by Western blotting for expression of M9TH-Fis1p (9 out of 16 positive). This strain displayed wild-type morphology in 64% of cells and moderate defects in the remaining cells. The same strategy was used to generate M3TH-FIS1 from the pRS403-3XMyc/TEV/URA3/TEV/His8 template (EG957) for subsequent experiments in the S288C background. This strain (DCY2192) displayed wild-type morphology in 89% of cells and mild defects in the remaining cells. DCY2192 was crossed to mdv1 and caf4
strains (Open Biosystems MATa deletion library) and sporulated to generate M3TH-FIS1 mdv1
(DCY2302) and M3TH-FIS1 caf4
(DCY2305).
fzo1::HIS5 was generated by transformation with a HIS5 (Saccharomyces kluyveri) fragment amplified with the FZO1 targeting primers Eg9 and Eg10. mito-GFP was integrated to the leu20 locus by transformation with NarI-digested EG686 (pRS403 + GPD/mito-GFP). mito-DsRed was integrated to the leu2
0 locus by transformation with HpaI-digested EG823 (pRS403 + GPD/mito-DsRed). dnm1
::HIS5 was generated by transformation with a HIS5 (S. kluyveri) fragment amplified with the DNM1-targeting primers Eg57 and Eg58.
Chromosomal CAF4-HTM was generated by transformation of DCY1979 with a His8/2TEV/9XMyc/HIS5 cassette (Seol et al., 1999) amplified with the CAF4 targeting primers Eg284 and Eg285. Chromosomal MDV1-HTM was generated transformation with the same cassette amplified with MDV1 targeting primers Eg80 and Eg81. Both CAF4-HTM and MDV1-HTM are functional because 70% of CAF4-HTM mdv1 yeast display spread mitochondrial nets and 95% of MDV1-HTM yeast cells display wild-type mitochondrial morphology.
DNM1-GFP was generated by amplifying GFP/HIS5 from pKT128 (Sheff and Thorn, 2004) with Eg342 and Eg343. This product was transformed into DCY1626 (wild-type yeast with mito-DsRed) to generate DCY2370. DCY2370 was crossed to fis1 and mdv1
caf4
strains to generate DCY2404 (DNM1-GFP fis1
), DCY2414 (DNM1-GFP caf4
), DCY2417 (DNM1-GFP mdv1
), and DCY2418 (DNM1-GFP mdv1
caf4
).
Tandem affinity purification MudPIT
Pellets from 2-l cultures (OD600 1.5) grown in YPD were prepared essentially as described previously for HPM tag Dual-Step affinity purification (Graumann et al., 2004), with the following modifications. Fungal protease inhibitors were used (Sigma-Aldrich) and lysates were cleared at 20 kg for 15 min. Cleavage from 9E10 beads was performed with GST-TEV protease for 3 h at RT. The second affinity step was performed with 40 µl Magne-His beads (Promega). Samples were proteolytically digested and analyzed by multidimensional chromatography in-line with a Deca XP ion trap mass spectrometer (ThermoElectron) as described previously (Mayor et al., 2005). Samples were released stepwise from the strong cation exchanger phase of the triphasic capillary columns as reported previously (Graumann et al., 2004).
Immunoprecipitation
CAF4-HA (EG1041), CAF4-HA residues 1274 (EG1043), and CAF4-HA residues 275659 (EG1045) were expressed in strains DCY1979 (wild-type) and DCY2305 (M3TH-FIS1 caf4). MDV1-HA (EG1047), MDV1-HA residues 1300 (EG1049), and MDV1-HA residues 301714 (EG1051) were expressed in DCY1979 or DCY2302 (M3TH-FIS1 mdv1
). Cultures were grown in selective SD media and harvested at OD600
0.8. 20 OD600 units of cells were lysed with glass beads (40 s with a vortex mixer, 4 times) in 500 µl ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, and 0.2% Triton X-100) in the presence of Fungal protease inhibitors (Sigma-Aldrich). Lysates were cleared by centrifuging 5 min at 5 krpm and 15 min at 14 krpm. At this point, a total lysate sample was taken. 400 µl of cleared lysate was mixed with a 20-µl bead volume of 9E10-conjugated protein ASepharose beads (Sigma-Aldrich) for 90 min. Beads were washed four times with 1-ml washes of lysis buffer. Precipitate was eluted with 100 µl SDS buffer at 95°C for 5 min. SDS-PAGE Western blotting was performed with 9E10 hybridoma supernatant (anti-Myc) or 12CA5 ascites fluid (anti-HA).
Yeast two-hybrid assay
pGAD vectors were transformed into PJ69-4. pGBDU vectors were transformed into PJ69-4a (James et al., 1996). Indicated vectors were mated on YPD plates using two transformants for each vector (totaling four matings per combination). Diploids were selected by replica plating to SD-leu-ura plates. Interactions were assayed by replica plating to SD-leu-ura-lys-ade and incubating for 4 d at 30°C.
Mitochondrial morphology analysis
Mitochondrially targeted GFP (mito-GFP) was used to monitor mitochondrial morphology. DCY1979 (wild-type), DCY1945 (caf4), DCY1984 (caf4
mdv1
), DCY2009 (fis1
), DCY2128 (mdv1
), DCY2155 (mdv1
dnm1
), and DCY2312 (dnm1
) were grown overnight, diluted 1:20 into fresh medium, and grown for 4 h at 30°C. Cultures were fixed by adjusting cultures to 3.7% formaldehyde and incubated 10 min at 30°C. Cells were washed 4 times with 1 ml PBS and scored for mitochondrial morphology. For CAF4 overexpression studies, plasmids p416 GalL/CAF4-HA (EG1133), p416 GalL/CAF4-HA residues 251659 (EG1135), or p416 GalL/CAF4-HA residues 1250 (EG1137) were transformed into DCY1979. Cultures were grown overnight in selective SRaff and diluted 1:20 in fresh YPD or YPGal and grown 3 h at 30°C. Samples were taken for Western analysis and the remaining culture was fixed as described above.
For latrunculin A treatment, overnight YPD cultures were diluted 1:20 in fresh YPD and grown for 3 h. Cultures were then treated for 1 h at 30°C with 200 µM latrunculin A in or with an equivalent amount of vehicle (DMSO). Cultures were then fixed as described above.
For time-lapse imaging, overnight SGal cultures were diluted 1:20 in fresh YPGal and grown for 3 h. Cells were pelleted, resuspended in fresh media, and embedded in 1% low melting point agarose containing 200 µM latrunculin A.
Bypass suppression assay
DCY2002 and DCY2343 were sporulated and dissected onto YPD plates. Spores were picked, grown overnight in 3 ml YPD at 30°C, pelleted, and resuspended to OD600 1.0 in YP. 3 µl of 1:5 serial dilutions were spotted on YPD and YPGlycerol and grown at 30°C for 2 and 4 d, respectively, to determine the fraction of cells that grow on glycerol. Genotypes were determined by PCR.
Differential centrifugation
Yeast strains CAF4-HTM (DCY2055), CAF4-HTM fis1 (DCY2094), MDV1-HTM (DCY2053), and MDV1-HTM fis1
(DCY2097) were grown in YPD and harvested at OD600
1.2. 100 OD units of cells were spheroplasted with zymolyase and lysed in a small clearance Dounce homogenizer (0.6 M sorbitol and 10 mM Tris, pH 7.4). The lysate was spun twice at 2.9 krpm for 5 min. An aliquot of the second supernatant was saved as the total lysate sample. The second supernatant was spun at 10 krpm for 10 min, and an aliquot of the supernatant was saved as the cytosol sample. The pellet was resuspended and spun again at 10 krpm for 10 min. An aliquot of the final pellet was saved as the mitochondrial pellet. Equal cell equivalents were loaded for Western blot analysis. The difference in porin intensity between the total and mitochondrial fractions most likely results from fewer obscuring proteins in the mitochondrial fraction.
Imaging
Images were acquired on a microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) using a 100x Plan-Apochromat, NA 1.4, oil-immersion objective. Z-stack images (between 0.1- and 0.2-µm intervals for still images and between 0.3- and 0.4-µm intervals for time-lapse images) were collected at RT with an ORCA-ER camera (Hamamatsu), controlled by AxioVision 4.2 software. Images were collected at either 30- or 40-s intervals for 30 min for time-lapse experiments. Iterative deconvolutions were performed with Axiovision 4.2 and maximum intensity projections were generated with AxioVision 4.2 for still images and Image J for time-lapse images. Fluorescent images in Figs. 35 were overlaid with differential interference contrast images (set at 50% opacity) in Adobe Photoshop CS.
Immunofluorescence
Cells were processed for immunofluorescence essentially as described previously (Guthrie and Fink, 1991) with the following modifications. Cultures were fixed for 15 min with 3.7% formaldehyde. Tween 20 (0.5%) was included in blocking buffer (PBS, 1% BSA) during a 15-min block step. Cells were stained with 9E10 hybridoma supernatant and a Cy3-conjugated antimouse secondary antibody. Washes after primary and secondary incubations were 5 min with blocking buffer, 5 min with blocking buffer containing 0.5% Tween 20, and two 5-min washes with blocking buffer. All incubations were performed at RT. GelMount (Biomeda) was used as mounting medium to preserve fluorescence.
Online supplemental material
Table S1 lists proteins identified in MudPIT experiments with M9TH-Fis1p. Table S2 shows yeast strains. Table S3 lists primer sequences. Videos 1 and 2 show mitochondrial fission in mdv1 yeast. Mitochondria were monitored by the mitochondrial outer membrane marker OM45-GFP. Arrows highlight a subset of fission events. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200503148/DC1.
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Acknowledgments |
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E.E. Griffin was supported by a National Institutes of Health training grant (NIH GM07616) and a Ferguson fellowship. MudPIT analysis was performed in the mass spectrometry facility of the laboratory of R.J. Deshaies (Investigator, Howard Hughes Medical Institute, Caltech). This facility is supported by the Beckman Institute at Caltech and by a grant from the Department of Energy to R.J. Deshaies and B.J. Wold. J. Graumann is supported by R.J. Deshaies through Howard Hughes Medical Institute funds. D.C. Chan is a Bren Scholar and Beckman Young Investigator. This research was supported by the National Institutes of Health (GM62967).
Submitted: 25 March 2005
Accepted: 9 June 2005
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References |
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