Cardiolipin and Mitochondrial Phosphatidylethanolamine Have Overlapping Functions in Mitochondrial Fusion in Saccharomyces cerevisiae*

Background: Cells lacking both cardiolipin and mitochondrial phosphatidylethanolamine are inviable, suggesting that these lipids have overlapping functions. Results: The loss of both lipids leads to decreased mitochondrial fusion and fragmented mitochondria. Conclusion: One overlapping function of these lipids is in mitochondrial fusion. Significance: Decreased mitochondrial fusion may partly explain the variation in clinical presentation observed in Barth syndrome. The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1Δpsd1Δ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TETOFF promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1⌬psd1⌬ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TET OFF promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.
Mitochondria exist as dynamic, double membrane-bound organelles. Mitochondrial membranes are enriched in phospholipids and proteins that are required for mitochondrial bio-genesis and for maintenance of mitochondrial morphology and the tubular network (1). CL 3 and PE are non-bilayer forming phospholipids in the mitochondrial membranes (2, 3) that play an essential role in mitochondrial function. Although cells lacking CL or mitochondrial PE are viable, the loss of both phospholipids is lethal, suggesting that these lipids have overlapping functions that are essential (4). Several recent studies have implicated the involvement of CL and mitochondrial PE in the maintenance of mitochondrial morphology (5)(6)(7). CL and PE are fusogenic phospholipids that form hexagonal phases in the presence of divalent cations, which confer negative curvature to the mitochondrial membrane (8,9). In the current study, we investigated the role of CL and PE in mitochondrial fusion.
Highly conserved protein machinery strictly regulates the process of mitochondrial fusion, and recent studies suggest that phospholipids also play a vital role in this process. Mitochondrial fusion in the yeast Saccharomyces cerevisiae primarily requires three proteins. These include the outer membrane GTPase, Fzo1p (Mfn1 and Mfn2 in mammals) (10,11), the inner membrane GTPase, Mgm1p (Opa1 in mammals) (12,13), and the outer membrane protein Ugo1p, which links the two GTPases to form a functional complex (14 -16). In S. cerevisiae, Mgm1p exists as long (l-Mgm1p) and short isoforms (s-Mgm1p), both of which are required for mitochondrial fusion (17,18). In vitro studies demonstrated that CL stimulates the GTPase activity of the s-Mgm1p (19,20). Moreover, it was shown in vitro that s-Mgm1p and l-Mgm1p assemble in a CLdependent manner (20). We hypothesized that the mitochondrial phospholipids CL and PE have overlapping functions in mitochondrial fusion in vivo. Consistent with this hypothesis, we demonstrate that cells lacking both CL and mitochondrial PE have reduced levels of both Mgm1p isoforms and exhibit excessive fragmentation of mitochondria and defects in mitochondrial fusion.

EXPERIMENTAL PROCEDURES
Yeast Strains, Plasmids, and Growth Media-The S. cerevisiae strains used in this study, listed in Table 1, are isogenic to BY4741 and BY4742. The single mutants were obtained from the MATa yeast deletion collection obtained from Dr. John Lopes. Double and triple mutants used in this study were obtained by tetrad dissection. Synthetic complete media contained standard concentration of amino acids, all the essential components of Difco vitamin-free yeast nitrogen base, 0.2% ammonium sulfate, and glucose (2%). Synthetic drop-out media contained all of the aforementioned ingredients except the amino acid was used as a selectable marker. Complex media contained yeast extract (1%), peptone (2%), with glucose (2%) (YPD) or galactose (2%) (YP-galactose) as a carbon source. All of the plasmids were amplified and extracted using standard protocols. The plasmids were transformed into yeast strains using a one-step transformation protocol (21). The v5 epitope-tagged CRD1 gene was cloned into the pCM189 plasmid (ATCC), in which, the TET OFF promoter regulates the expression of cloned gene, using the BamHI and NotI restriction sites. The existing URA3 marker of the plasmid was replaced by HIS3 using EcoRV and ClaI restriction sites. Bacterial transformations were performed using dam Ϫ E. coli to avoid Dam methylase sensitivity to the ClaI restriction enzyme.
Fluorescence Microscopy-Fluorescence microscopy was performed using an Olympus BX41 epifluorescence microscope. Images were acquired using an Olympus Q-Color3 digitally charge-coupled device camera operated by QCapture2 software. All pictures were taken at 1000ϫ. To stain mitochondrial DNA, yeast cells were cultured to the mid-log phase, fixed in 70% ethanol at room temperature for 30 min, washed two times with distilled water, and stained with 1 g/ml DAPI (Sigma) for 5 min. Mitochondria were visualized by transforming the cells with either plasmid pYX142 or pYX122 expressing GFP fused to the mitochondrial presequence, pre-Su9 (22) (provided by Dr. Benedikt Westermann) or pYX142-mtRFPexpressing mitochondria-targeted RFP (provided by Dr. Janet Shaw). Cells were harvested in the appropriate medium and viewed under fluorescence microscopy.
Electron Microscopy-Cells were grown in 100 ml of YPD to an A 550 of 0.5. After harvesting, cells were prepared for EM using the osmium thiocarbohydrazide osmium fixation method (23).
In Vivo Fusion Assay-The mitochondrial in vivo fusion assay was performed as described (13,24). MAT␣ cells of WT, crd1⌬, and psd1⌬, were transformed with pYX122-mtGFP and MATa cells were transformed with pYX142-mtRFP. MATa cells of the conditional mutant crd1⌬psd1⌬ were transformed with pYX142-mtRFP, and MAT␣ cells were transformed with pYX142-mtGFP. MATa cells of the conditional mutant crd1⌬psd1⌬fis1⌬ were transformed with pYX142-mtGFP, and MAT␣ with pYX142-mtRFP. MAT␣ cells of crd1psd1dnm1⌬ were transformed with pYX142-mtGFP and MATa with pYX142-mtRFP. Cells were grown in 5 ml of selective media to an A 550 of 0.5. After centrifugation, cells of opposite mating type were mixed and spotted on an YPD plate. After 3.5 h of incubation at 30°C, cells were observed for mitochondrial fusion. The images were merged and analyzed using Image J software.
Extraction, Separation, and Analysis of Yeast Total Phospholipids-Yeast cells were grown in the presence of 32 P i (10 Ci/ml) in the indicated growth conditions. Total phospholipids were extracted and analyzed by TLC as described (25). The developed chromatograms were analyzed by phosphorimaging, and the phospholipids were quantified using Image Quant software.
Flow Cytometry-Mitochondrial membrane potential was measured using whole cells as described (26). Cells were grown in YP-galactose media to the mid-logarithmic phase. Actively growing cells (5 ϫ 10 4 cells) were incubated at 30°C with the dye tetramethyl rhodamine methyl ester (TMRM) (50 nM) for 30 min. To induce a decrease in membrane potential, control cells were treated with sodium azide (20 mM). Fluorescence was measured using a flow cytometer. The results were analyzed using WinMDI2.9 software.

Maintenance of Mitochondrial Network and Mitochondrial Fusion Is Defective in Absence of CL and Mitochondrial PE-
Previous studies have shown that loss of CL (crd1⌬) is lethal in combination with loss of mitochondrial PE (psd1⌬), but not cytosolic PE (psd2⌬) (4). To gain insight into the overlapping roles of these mitochondrial lipids, we constructed a conditional mutant, crd1⌬psd1⌬, in which CRD1 is expressed from a plasmid under the control of the TET OFF promoter. This mutant lacks mitochondrial PE and CL in the presence of tetracycline but contains CL in the absence of tetracycline. We used this conditional mutant as a tool to identify functions of these phospholipids in mitochondrial morphology and mitochondrial fusion. The conditional double mutant grew normally on YPD. The addition of tetracycline (200 g/ml), which shut off CRD1 expression, inhibited growth of the double mutant but did not affect growth of WT, crd1⌬, or psd1⌬ cells (Fig. 1A). To determine whether tetracycline did indeed regulate CRD1 expression, we measured the levels of CL in crd1⌬psd1⌬ cells. In psd1⌬, CL was synthesized, although levels were reduced compared with those of WT, consistent with previ-ous studies (4). In crd1⌬psd1⌬ grown in the absence of tetracycline, CL levels were 40% of those of psd1⌬, indicating that CL levels from plasmid CRD1 are less than CL levels obtained from genomic CRD1. In the presence of tetracycline, CL was greatly diminished to only 14% of the levels in psd1⌬, indicating that expression from the TET OFF promoter was greatly (but not completely) repressed. Tetracycline itself did not affect CL levels in cells lacking the plasmid, which were similar in psd1⌬ cells grown in the presence and absence of the drug (Fig. 1B).
To determine whether CL and mitochondrial PE play a role in the maintenance of mitochondrial morphology, we compared the mitochondrial network in WT, crd1⌬, psd1⌬, and crd1⌬psd1⌬ cells transformed with plasmids expressing mitochondria targeted GFP (mtGFP) ( Fig. 2A) (22). At least 500 cells of each strain were observed for each biological replicate (Fig.  2B). Cells exhibited a normal tubular mitochondrial network in crd1⌬, consistent with earlier findings (27), indicating that the lack of CL by itself does not affect the mitochondrial network. The lack of mitochondrial PE had a small but significant effect on the mitochondrial network, as ϳ23% of psd1⌬ cells exhibited fragmented mitochondria. Unlike the WT-like tubular mitochondrial network, the majority of psd1⌬ cells had short tubular mitochondria consistent with a mitochondrial morphology defect in these cells. The morphology of crd1⌬psd1⌬ cells grown in the absence of tetracycline was similar to that of psd1⌬ cells. However, the addition of tetracycline severely affected the mitochondrial network, leading to excessive mitochondrial fragmentation similar to that observed in fusion mutants (Fig. 2, A and B). These findings suggested that loss of both CL and mitochondrial PE leads to a defect in mitochondrial fusion. Tetracycline by itself did not affect the mitochondrial network in WT, crd1⌬, and psd1⌬ cells (data not shown).
To determine whether the increase in mitochondrial fragmentation correlated with the loss of CL, a time course experiment was performed in which crd1⌬psd1⌬ cells were grown in YPD containing 32 P i , in the presence or absence of tetracycline. Total phospholipids and mitochondrial morphology were examined at 5, 8, and 11 h. Total CL decreased by ϳ11, ϳ31, and ϳ55%, whereas the percentage of mitochondrial fragmentation increased during this time to ϳ20, ϳ45, and ϳ96% at 5, 8, and 11 h, respectively (Fig. 2, C and D). These findings indicate that the increase in mitochondrial fragmentation corresponded with a decrease in CL in the crd1⌬psd1⌬ cells.
Electron microscopic examination of the mutants revealed that crd1⌬ mitochondria were somewhat smaller than those of WT but relatively unremarkable (data not shown). Mitochondria in psd1⌬ cells and in crd1⌬psd1⌬ cells grown in the absence of tetracycline also appeared smaller than WT. This phenotype was more significant in crd1⌬psd1⌬ cells grown in the presence of tetracycline. Thus, the loss of both CL and mitochondrial PE led to highly fragmented mitochondria, consistent with defective fusion (Fig. 2F).
To determine the role of CL and mitochondrial PE in mitochondrial fusion, we performed an in vivo fusion assay (13,24) as described under "Experimental Procedures." In this assay, we examined mitochondrial fusion events in zygotes acquired by mating haploids of opposite mating types of WT, crd1⌬, psd1⌬, and crd1⌬psd1⌬ cells transformed with either mtGFP or mitochondria-tagged RFP (mtRFP). As expected, crd1⌬ cells exhibited complete mixing of mitochondrial content, indicating that the lack of CL alone does not affect mitochondrial fusion (Fig.  3A). Fusion occurred but was decreased in psd1⌬ cells, suggesting that the lack of PE causes somewhat reduced fusion even when CL is present. As expected, the fusion phenotype of crd1⌬psd1⌬ cells grown in the absence of tetracycline was similar to that of psd1⌬ cells. However, in the presence of tetracycline, a complete block of mitochondrial fusion was observed in all the crd1⌬psd1⌬ zygotes examined, consistent with the defective mitochondrial network observed in the absence of both CL and mitochondrial PE (Fig. 3A). These results indicate that when both CL and mitochondrial PE are deficient, mitochondrial fusion does not occur.
Loss of Mitochondrial DNA and Reduced Mitochondrial Membrane Potential in Cells Lacking CL and Mitochondrial PE-Several studies have reported that cells defective in mitochondrial fusion lose mitochondrial DNA (mtDNA) (10,18,24,28,29). Therefore, we hypothesized that crd1⌬psd1⌬ cells would exhibit mtDNA loss. To address this possibility, WT, crd1⌬, psd1⌬, and crd1⌬psd1⌬ cells were grown with or without tetracycline to the mid-logarithmic growth phase at 30°C. Cells were observed under the fluo-rescence microscope after DAPI staining for the presence of mtDNA (Fig. 4A). As expected, the majority of crd1⌬psd1⌬ cells (ϳ80%) grown in the absence of tetracycline at the permissive temperature of 30°C retained mtDNA. This was consistent with our previous study showing that crd1⌬ cells retained mtDNA at 30°C but exhibited mtDNA loss only at elevated temperatures (30). However, in the presence of tetracycline, only ϳ20% of crd1⌬psd1⌬ cells had mtDNA (Fig.  4B).
Mitochondrial fusion as determined by in vitro assay involves distinct steps of outer and inner membrane fusion (31). In addition to functional protein complexes, fusion of the outer membrane requires low GTP levels and a proton gradient, whereas inner membrane fusion requires large amounts of GTP and an inner membrane potential. It is therefore possible that a decreased membrane potential led to the fusion defect in crd1⌬psd1⌬ cells. To test this possibility, we used a flow cytometry assay to measure mitochondrial membrane potential (⌬⌿ m ) in intact WT, crd1⌬, psd1⌬, and crd1⌬psd1⌬ cells grown with or without tetracycline (26) in YP-galactose rather than YP-glucose to ensure actively respiring mitochondria. Cells were grown at 30°C to the mid-logarithmic growth phase and then incubated with the voltage-dependent probe TMRM (50 nM) for 30 min. The accumulation of TMRM in mitochon- dria is driven by the ⌬⌿ m , which is determined by the difference in yellow fluorescence and forward scatter in the form of fluorescence peaks (26). Values were calculated relative to the control, i.e. crd1⌬psd1⌬ cells grown in the absence of tetracycline. As seen in Fig. 4C, crd1⌬psd1⌬ cells in the presence of tetracycline exhibited a decrease in membrane potential similar to that observed in these cells in the presence of sodium azide, a cytochrome c oxidase inhibitor that reduces the ⌬⌿ m (26). These observations were consistent with a reduced membrane potential in cells lacking both CL and mitochondrial PE. It was recently demonstrated that mitochondrial fusion in mammalian cells requires high ⌬⌿ m levels and is prevented by depolarization (32). Thus, the observed decrease of ⌬⌿ m could be one explanation for the fusion defects in the crd1⌬psd1⌬ mutant cells.

Deletion of DNM1 in crd1⌬psd1⌬ Cells Restores Normal
Mitochondrial Tubular Network-We wished to determine whether the mitochondrial fragmentation observed in crd1⌬ psd1⌬ cells could be explained by increased fission rather than decreased fusion. Fusion and fission regulate mitochondrial morphology in an antagonistic manner (33). Previous studies have shown that three major proteins regulate mitochondrial fission, Dnm1p (34 -36), Fis1p (36), and Mdv1p (37,38). Abolishing mitochondrial fission by deletion of any of these genes leads to net-like mitochondria. In contrast, eliminating fusion by deletion of MGM1, FZO1, or UGO1 leads to fragmentation, which can be restored to normal tubular morphology by deletion of the fission gene DNM1 (13,35). If mitochondrial fragmentation in crd1⌬psd1⌬ cells results from a defect in fusion and not increased fission, then disruption of mitochondrial fis- sion would restore mitochondria to the normal tubular morphology. Therefore, we examined whether the fragmented mitochondrial morphology of crd1⌬psd1⌬ cells could be rescued to normal tubular mitochondrial morphology by deletion of the fission gene DNM1. To do so, we constructed a crd1⌬ psd1⌬dnm1⌬ conditional mutant containing the plasmid with the TET OFF -regulated CRD1 expression plasmid, as well as a plasmid expressing mtGFP (Fig. 5A). In the absence of tetracycline, when CRD1 is expressed, the crd1⌬psd1⌬dnm1⌬ cells would be expected to exhibit net-like mitochondria characteristic of a fission defect. However, in the presence of tetracycline, the cells would be predicted to lack both fission and fusion and, hence, would exhibit WT tubular mitochondrial morphology.
As seen in Fig. 5B, ϳ76% of crd1⌬psd1⌬dnm1⌬ cells grown in the absence of tetracycline exhibited net-like mitochondria, the predicted phenotype. The remaining cells (ϳ18%) exhibited tubular mitochondria, most likely because fusion was decreased in these cells due to the low level of expression of CRD1 (Fig. 1). In the presence of tetracycline, only ϳ32% of cells exhibited net-like mitochondria, whereas the majority (ϳ45%) exhibited tubular mitochondria, as predicted. These findings suggest that both fission and fusion were defective in these cells and that the fragmented mitochondrial morphology in crd1⌬psd1⌬ cells was rescued by deletion of the fission gene DNM1 (Fig. 5, A and B). Tetracycline itself did not affect the mitochondrial morphology in dnm1⌬ (data not shown). Interestingly, a significant number of crd1⌬psd1⌬dnm1⌬ cells (ϳ22%) grown in the presence of tetra-cycline, had fragmented mitochondria, as the network exhibited the appearance of a string of beads (data not shown). This morphology suggested the presence of a persistent fusion defect in the absence of CL and mitochondrial PE.
To further investigate the block in fusion, we performed an in vivo mitochondrial fusion experiment by mating crd1⌬psd1⌬ dnm1⌬ cells of opposite mating types, in which one mating type contained mtGFP and the other mating type contained mtRFP. We observed decreased fusion in cells grown without tetracycline, and a complete block in mitochondrial fusion in cells grown with tetracycline (Fig. 5C). Cells grown without tetracycline that exhibited net-like structures had no defect in mitochondrial fusion (Fig. 5C). Cells grown in the presence of tetracycline displayed a complete block of mitochondrial fusion. Similar observations were made in the conditional mutant crd1⌬psd1⌬fis1⌬ (supplemental Fig. S1). These experiments suggest that crd1⌬psd1⌬dnm1⌬ cells exhibited a fusion defect due to loss of CRD1 and PSD1. Taken together, these studies indicate that mitochondrial fragmentation observed in crd1⌬psd1⌬ cells is a result of defective fusion and not due to increased fission.
To determine whether deletion of the fission gene FIS1 or DNM1 could rescue the lethality of the double mutant, we crossed crd1⌬dnm1⌬ and crd1⌬fis1⌬ with psd1⌬ and carried out meiotic tetrad analysis to identify viable triple mutants. However, triple mutants were not detected in 72 tetrads of the  diploid crd1⌬fis1⌬PSD1/CRD1FIS1psd1⌬ or 75 tetrads of the diploid crd1⌬dnm1⌬PSD1/CRD1DNM1psd1⌬. Therefore, although CL and mitochondrial PE have overlapping functions in mitochondrial fusion, rescue of the fusion defect could not rescue the synthetic lethality.
Reduced Steady State Levels of l-Mgm1 and s-Mgm1 Isoforms in Cells Lacking CL and Mitochondrial PE-The current study suggests that one common function of CL and PE is mitochondrial fusion. It has been reported that the lack of CL destabilizes the anchoring, assembly, and GTPase activity of fusion protein Mgm1p in vitro (19,20,39). To test whether mitochondrial PE compensates for the loss of CL and stabilizes the fusion proteins in vivo, we determined the steady state levels of fusion proteins Fzo1p, Ugo1p, l-Mgm1p, and s-Mgm1p in WT, crd1⌬, psd1⌬, and crd1⌬psd1⌬ cells. The crd1⌬psd1⌬ cells exhibited significantly diminished levels of l-Mgm1p and s-Mgm1p (Fig. 3, B and C). Fzo1p levels were slightly decreased and Ugo1p was not affected (Fig. 3, B and C). To determine whether the loss of Mgm1p isoforms correlated with the loss CL in crd1⌬psd1⌬, cells were grown in the presence or absence of tetracycline, proteins were extracted from cells harvested at 5, 8, and 11 h, and the levels of Mgm1p isoforms were determined by Western blot (Fig. 2E). The isoform levels were severely diminished at 11 h, which correlated with increased mitochondrial fragmentation as seen in Fig. 2C. These data indicate that the defect in mitochondrial fusion in crd1⌬psd1⌬ can be attributed at least in part to the reduced levels of s-Mgm1p and 1-Mgm1p.

DISCUSSION
In this study, we demonstrate that crd1⌬psd1⌬ cells lacking both CL and mitochondrial PE have fragmented mitochondria due to a defect in mitochondrial fusion. In addition to this defect, we show that crd1⌬psd1⌬ cells exhibit loss of mtDNA, decreased membrane potential, and reduced steady state levels of short and long isoforms of Mgm1p, a mitochondrial inner membrane protein essential for fusion. The fragmented mitochondrial morphology along with the fusion defect observed in crd1⌬psd1⌬ cells were rescued by deletion of the fission genes DNM1 or FIS1. These data indicate that CL and mitochondrial PE are required for mitochondrial fusion in vivo.
Our previous studies have shown a synthetic lethal interaction between crd1⌬ and psd1⌬ mutant cells, suggesting essential overlapping roles of CL and mitochondrial PE (4). PE synthesized by the non-mitochondrial pathway (Psd2p catalyzed PE synthesis in Golgi/vacuole) (40 -42) did not rescue this lethality. Externally synthesized PE is inefficiently transported to the inner mitochondrial membrane, as reduced levels of PE were observed in the inner mitochondrial membrane of the psd1⌬ mutant cells (43). Taken together, these studies suggested that PE synthesized in the mitochondrial inner membrane has functions that cannot be compensated by externally synthesized PE. In the current study, we demonstrate that the loss of mitochondrial phospholipids CL and PE leads to mitochondrial fragmentation (Fig. 2, A, B, and F) and defective mitochondrial fusion (Fig. 3A). Although mitochondrial fusion is an overlapping function of CL and PE, the lack of mitochondrial fusion is probably not the cause of lethality observed in crd1⌬psd1⌬ cells, as lethality was not rescued by deletion of the fission gene FIS1 or DNM1. Mitochondria are required not only for cellular bioenergetics, but also for the synthesis of essential metabolites. In addition, our previous studies have shown that CL is required for non-mitochondrial functions, including vacuolar function, the high osmolarity glycerol (44) pathway, and cell wall synthesis (45)(46)(47). Thus, it is possible that lethality in cells lacking CL and PE could be caused by deficiencies in both mitochondrial and non-mitochondrial functions. The identification of suppressors of crd1⌬psd1⌬ synthetic lethality will very likely identify the essential cellular functions shared by these phospholipids. These studies are currently in progress.
How do CL and mitochondrial PE affect mitochondrial fusion? Non-bilayer lipids are known to affect the function and stability of many mitochondrial membrane proteins (48). Recent studies have proposed that scaffolding proteins such as prohibitin recruit membrane proteins to CL-and PE-rich regions, forming protein-rich lipid domains (49). The lack of CL and mitochondrial PE might influence the distribution of these domains, which in turn, would affect several mitochondrial processes, including mitochondrial fusion. Although early studies suggested that the non-bilayer forming phospholipids CL and PE play an important role in mitochondrial fusion, very little was known about the mechanism by which this could occur (8,50,51). In this study, we show that the lack of CL and mitochondrial PE leads to reduced steady state levels of both large and small isoforms of Mgm1p (Figs. 2E and 3B), which are required for fusion. Recent studies have shown that l-Mgm1p acts as an anchor in the inner membrane (17). Both CL and PE are synthesized and predominantly localized in the inner mitochondrial membrane, and the loss of both CL and mitochondrial PE might affect the stability of this isoform, leading to its degradation. The formation of s-Mgm1p requires functional mitochondrial protein import machinery, membrane potential and adequate ATP levels, all of which are defective in cells lacking CL (52)(53)(54)(55)(56). This is a first report describing overlapping roles of CL and mitochondrial PE in fusion in vivo and suggests a mechanistic role for these phospholipids in regulating mitochondrial structure and function.
How is the role of CL and PE in mitochondrial fusion relevant to human disease? The role of mitochondrial phospholipids in fusion is relevant to studies that implicate function of mitochondrial fusion in cardiac function (57). Fragmented mitochondria are associated with the loss of Opa1 (the human homolog of Mgm1p) in mitochondrial myopathies involving cardiac and skeletal muscle (53) and in ischemic cardiomyopathy (58). Overexpression of the fusion proteins Mfn1/2 (human homolog of Fzo1p) prevents cardiac cell death from ischemia (59). Elucidating the role of CL and PE in mitochondrial fusion may also shed light on defects observed in lymphoblast mitochondria from patients with Barth syndrome (BTHS), a severe genetic disorder characterized by dilated cardiomyopathy and skeletal myopathy (60,61). BTHS is caused by mutation in the CL remodeling enzyme tafazzin, resulting in decreased CL and altered fatty acid composition of major mitochondrial phospholipids, including CL and PE (62). Defects in mitochondrial fusion may account for the observed morphological variation in BTHS mitochondria, including enlarged size, fragmentation, adhesion of opposing membranes, and deformed intercristae space observed in BTHS lymphoblasts as well as in cardiac and skeletal muscle mitochondria of the mouse model of BTHS (63,64). Identifying the role of CL and PE in mitochondrial fusion may thus explain, in part, the wide variation in the clinical presentation observed in BTHS.