Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.

The endoplasmic reticulum (ER) forms membrane contact sites (MCS) with several different organelles, including mitochondria, peroxisomes, the Golgi apparatus, endosomes and the plasma membrane (1). These sites play critical roles in cell signaling, lipid homeostasis and apoptosis (2,3). Recent studies show that contact sites play a structural role in maintaining the normal morphology of the interacting organelles. In addition, ER-mitochondrial contact sites can be maintained as organelles move within a mammalian cell (4).
Several different proteins have been implicated in tethering the ER to mitochondria in mammals and budding yeast (2,5). One of the best-characterized tethers is the ER Mitochondria Encounter Structure (ERMES), which links the ER to the outer mitochondrial membrane in Saccharomyces cerevisiae (6). ERMES is composed of four proteins that associate to form a complex. Mmm1 is anchored in the ER and Mdm10 in the outer mitochondrial membrane (6,7), whereas the cytosolic protein Mdm12 and the mitochondrial-associated protein Mdm34 bridge the interaction between Mmm1 and Mdm10 in the complex (8,9). Using fluorescent markers, ERMES appears as multiple discrete puncta (8)(9)(10) at sites of ER-mitochondrial juxtaposition (6), suggesting that these four proteins assemble into higher order structures. In the absence of any single ERMES protein, the complex falls apart and puncta are no longer visible (6); while the morphology of the ER remains intact, the normally tubular mitochondrial network is converted into one or more large spheres (7,9,11,12).
The functional role of the ERMES complex is unclear. Mdm10 and Mdm12 were originally proposed to control mitochondrial shape and mitochondrial inheritance during cell division (7,11). Additional studies suggested that Mmm1, Mdm10 and Mdm12 attach mitochondria to the actin cytoskeleton for polarized transport to the growing yeast bud (8,13). The fact that ERMES mutants lose mitochondrial DNA (mtDNA) led to the suggestion that ERMES proteins control mitochondrial genome maintenance (9,10). Consistent with this idea, Mmm1 has been shown to co-localize with replicating mtDNA nucleoids (14). In addition, a link between actin filaments, ERMES proteins and mtDNA nucleoids appears to couple inheritance of mitochondrial membranes and genomes during division (8). Mdm10, Mmm1 and Mdm12 were also shown to function in the import and assembly of mitochondrial ß-barrel proteins (15)(16)(17). Finally, the ERMES complex has recently been linked to the transport of phosphatidylserine (PS) from the ER into mitochondria (6,18), where it can serve as a substrate for phosphatidylethanolamine (PE) synthesis (19,20). A role for ERMES in lipid transport is also supported by the fact that three ERMES proteins (Mmm1, Mdm12 and Mdm34) have SMP (synaptotagmin-like, mitochondrial and lipid-binding proteins) domains implicated in binding hydrophobic ligands, including lipids (21).
This bewildering list of potential functions of the ERMES complex raises the question of whether some of them are indirect. For example, it is conceivable that the deletion of ERMES components causes mitochondrial morphology or mitochondrial protein import defects, which in turn cause problems in other functions. Indeed, it was recently shown that mitochondrial morphology defects in some ERMES mutants are a secondary consequence of mitochondrial protein import defects (15).
New findings raise the possibility that ERMES is regulated by Gem1, a mitochondrial outer membrane protein in the conserved Miro (mitochondrial rho-like) GTPase family. Gem1 contains two GTPase domains and two calciumbinding EF-hand motifs, all of which are required for function (22). Correlation of GEM1 and all four ERMES loci in a genetic interaction map suggested that these genes are functionally related (6,18). Similar phenotypes have been described for gem1 and ERMES mutants, including abnormal mitochondrial morphology and loss of mtDNA (22). In addition, genetic studies have implicated Gem1 in mitochondrial inheritance during cell division (23) and regulation of lipid synthesis by ERMES (24). Finally, Gem1 was recently identified as a substoichiometric component of the ERMES complex (24,25).
Here, we show that the ERMES complex or Gem1 have no direct role in the transport of PS from the ER to mitochondria during the synthesis of PE. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes do not depend on the presence of Gem1 and are often long-lived. Taken together, our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.

Results
The ERMES complex and Gem1 do not directly affect phosphatidylserine transport from the ER to mitochondria The ERMES complex has been proposed to facilitate the transport of lipids between the ER and mitochondria by providing a physical link between the two organelles (6,24). Specifically, ERMES may promote the transfer of ER-synthesized PS to mitochondria. Once in mitochondria, PS is converted to PE by the phosphatidylserine decarboxylase Psd1, an enzyme located in the inner membrane (see scheme in Figure S1) (19,20). The reported genetic link between ERMES components and Gem1 raises the possibility that the Gem1 GTPase may also function in lipid transport (6,18).
We first addressed whether ERMES is involved in PS transport from the ER to mitochondria by following the conversion of PS to PE in vivo. Yeast cells were incubated with radioactive serine, the phospholipids were extracted, separated by thin-layer chromatography (TLC), and analyzed by autoradiography. These studies were performed in a strain that lacks the second phosphatidylserine decarboxylase Psd2, which is localized to the Golgi and also contributes to PE synthesis. Compared with the psd2 control, the percentage of PS converted to PE is not significantly reduced in mutants that lack both an ERMES component and Psd2 ( Figure 1A and S2 control). By contrast, the simultaneous absence of Psd1 and Psd2 resulted in a significant reduction ( Figure 1A). The small decrease observed for mdm34 and mdm10 could result from insufficient Psd1 in the mitochondria of these strains (see Figure S3A). Similar results were obtained in vitro. Crude isolated mitochondria that retain ER-mitochondria connections (26) were incubated with radiolabeled serine, and the conversion of the synthesized PS to PE was determined by TLC and autoradiography. While mitochondria lacking Psd1 were totally inactive, the deletion of ERMES components had no effect ( Figure 1B). Collectively, these data show that the ERMES components are not essential for the transfer of PS between the ER and mitochondria. Analysis of the total cellular phospholipid composition showed that PE and PC remained unchanged in mutants lacking both Psd2 and either Mmm1 or Mdm12, and decreased moderately in mutants lacking both Psd2 and either Mdm34 or Mdm10 ( Figure 1C). PS decreased moderately in the mutant lacking both Psd2 and Mmm1, increased moderately in the mutant lacking Mdm12 and remained unchanged in the other two double mutants. These results support the conclusion that the disruption of the ERMES complex does not dramatically compromise phospholipid metabolism.
Next, we asked whether Gem1 is required for PS to PE conversion. Using the in vivo assay, the conversion of PS to PE was only modestly altered by the absence of Gem1 (Figure 2A). Of particular significance is the finding that no defect in PS conversion to PE was observed when experiments were performed directly on crude mitochondria in vitro ( Figure 2B). We also compared the phospholipid composition in psd2 and psd2 gem1 strains. Quantification of total cellular phospholipids by TLC revealed only insignificant differences between the gem1 psd2 strain and a psd2 control ( Figure 2C). The phospholipid compositions of mitochondrial fractions A) The conversion of PS to PE was determined in vivo by incubating yeast cells with radioactive serine, extracting phospholipids, and analysis by TLC. The percentage of radiolabeled PS converted to PE in psd2 control and mutant strains is shown. Labeling of the different ERMES mutants was comparable with the exception of mdm10 , which consistently incorporated less label than psd2 control and the other mutants. B) The conversion of PS to PE was determined in vitro using crude mitochondria incubated with radioactive serine. The percentage of radiolabeled PS converted to PE in WT and mutant mitochondria is shown. Bars and error bars represent the average and SD from three independent experiments. C) Relative changes in phospholipid composition in ERMES mutants. Lipid extracts of the indicated strains were analyzed by shotgun lipidomics (35). The abundance of the indicated lipid classes are shown relative to the control strain psd2 . Samples were analyzed in duplicate and average values are shown. The maximal difference between two duplicate measurements was 1.2%. PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol.
isolated from these strains were also similar ( Figure 2D). These results argue against a specific role for Gem1 in regulating transport of PS into mitochondria, or a general role in phospholipid metabolism.

The ERMES complex does not play a direct role in mitochondrial inheritance
Next, we addressed how ERMES components affect mitochondrial inheritance (7,9,11,12). During cell division in WT yeast cells, 100% of all medium-and largesized buds inherit mitochondria from the mother cell ( Figure 3A). In contrast, in mdm12 or mmm1 single mutants, only 40 or 51% of medium and large buds contain mitochondria, respectively. This is an underestimate of the defect, since we score inheritance as successful if a large bud contains only one or a few mitochondrial fragments. Although mitochondrial inheritance defects have been reported for mdm10 , in our strain background, mdm10 derived from a newly dissected tetrad displayed essentially wild-type inheritance (though it grew slowly). The mdm10 strain contained short tubular mitochondria in addition to the round or globular mitochondria characteristic of mdm12 or mmm1 (unpublished observations). Owing to its lack of mitochondrial inheritance phenotype, the mdm10 strain was not analyzed further.
To test whether the observed mitochondrial inheritance defects are a secondary consequence of mitochondrial morphology defects, we overexpressed Ypt11 in the mmm1 and mdm12 strains. Ypt11 binds to the tail of the Myo2 motor protein, and its overexpression was previously shown to promote the transport of mitochondria out of the mother cell into the bud (23,27). When overexpressed in ERMES mutants, Ypt11 increased mitochondrial inheritance from 40 to 81% in mdm12 and from 59 to 89% in mmm1 cells ( Figure 3B). Importantly, overexpression of Ypt11 did not rescue mitochondrial morphology defects in the ERMES mutants; essentially 100% of mdm12 and mmm1 cells overexpressing Ypt11 retained mutant mitochondrial morphology ( Figure 3C). Thus, the morphology and inheritance defects in mdm12 and mmm1 mutants are not obligatorily linked.
To further test whether morphology defects cause inheritance defects, we used an artificial tether between ER and mitochondria (28), called ChiMERA (Construct helping in Mitochondria-ER Association) (6). As reported previously (6), ChiMERA expression restored tubular mitochondrial morphology in 73% of mdm12 cells ( Figure 3C). It also rescued the mitochondrial inheritance defect in this strain ( Figure 3B). By contrast, ChiMERA did not rescue either the mitochondrial morphology ( Figure 3B and (6)) or the inheritance defects in the mmm1 mutant ( Figure 3B). ChiMERA expression also did not rescue the mitochondrial morphology and inheritance defects observed in the gem1 mutant ( Figure S4, and unpublished observations). When considered together, our results demonstrate that:   (i) enlarged, spherical mitochondria in ERMES mutants can be transported from the mother cell to the bud during cell division, (ii) normal, tubular mitochondria are inherited more efficiently than morphologically distorted mitochondria and (iii) ERMES proteins are not essential components of the mitochondrial inheritance machinery.
We showed previously that growth defects in gem1 , ypt11 and mmr1 mutants are correlated with mitochondrial inheritance defects (23). (Like Ypt11, Mmr1 is a Myo2 adapter that promotes mitochondrial inheritance (29).) If Gem1 acts in the same or a parallel pathway with ERMES, we would expect growth defects and inheritance defects to correlate in ERMES mutants as well. However, this was not the case. As shown previously (6), ChiMERA expression rescued the growth defect of mdm12 (Figure 4), consistent with the observation that ChiMERA rescues inheritance defects in this strain ( Figure 3B). However, we found that overexpression of Ypt11 in mdm12 cells rescued mitochondrial inheritance defects, but not the growth defects ( Figures 3B and 4). Thus, the growth defect of the mdm12 ERMES mutant is not caused by a mitochondrial inheritance defect. By contrast, neither ChiMERA nor Ypt11 overexpression rescued growth defects in strains lacking the integral membrane proteins Mmm1 or Mdm10 ( Figure 4).

ERMES complex formation does not require GEM1, YPT11 or MMR1, genes implicated in mitochondrial inheritance
To further test the relationship between Gem1 and ERMES, we visualized the ERMES complex by tagging its ER component Mmm1 with GFP (Mmm1-GFP). Mitochondria were simultaneously imaged using mitochondrialtargeted RFP (mtRFP). Essentially all WT cells contained multiple ERMES puncta that colocalized with the tubular mitochondrial network ( Figure 5). Although previous studies reported that yeast cells contain one to ten ERMES complexes (8-10,24), we often observed more puncta, all of which aligned along the mitochondrial surface.
Our ability to visualize more ERMES puncta per cell is likely due to differences in imaging equipment and strain backgrounds used. In addition, a subset of Mmm1-GFP marked ERMES puncta underwent rapid photobleaching, and there may be additional ERMES assemblies that are too small to visualize by fluorescence microscopy. Thus, there are likely more ERMES complexes connecting the ER to mitochondria in vivo than previously appreciated. Importantly, in the absence of Gem1, 99% of the cells contained ERMES puncta despite the fact that the cells have globular and fragmented mitochondria. Thus, ERMES complexes assemble at ER-mitochondrial junctions even when mitochondrial morphology is abnormal, and the aberrant mitochondrial morphology in the gem1 mutant is not due to an inability to form ERMES puncta. ERMES puncta were also observed in mitochondrial inheritance mutants ypt11 and mmr1 , which were previously shown to have synthetic growth defects with gem1 (23). As shown in Figure 5, 96% of cells in mmr1 or ypt11 strains contained ERMES puncta. It should be noted that these cells have tubular mitochondria.
Immunoprecipitation experiments support the notion that Gem1 is not required for ERMES complex formation. When lysates from cells expressing functional Mmm1-GFP were subjected to immunoprecipitation with GFP antibodies, both Mdm10 and Mdm12 were coimmunoprecipitated ( Figure 6, lane 7). As expected, in the absence of Mdm12 or Mdm34, Mdm10 was no longer immunoprecipitated (lanes 8 and 9). Importantly, Mdm10 and Mdm12 remain associated when Gem1 is absent (lane 10). Together, these data indicate that Gem1 is not a major structural component of ERMES and is not essential for formation of the ERMES complex. In contrast to a previous report (24), the number and appearance of ERMES puncta was normal in strains over-expressing Gem1 or in strains expressing Gem1 proteins that carry mutations in the GTPase domain or EF-hand (unpublished observations).

The ERMES complex is a stable structure
Because our results suggested that the ERMES complex may be primarily involved in maintaining the structural integrity of mitochondria, we tested its stability by livecell imaging. We used fluorescence microscopy to follow Mmm1-GFP labeled ERMES complexes over time. Over the course of 40 minutes, many ERMES puncta remained stationary within a cell (Figure 7, arrows), while some appeared to migrate (Figure 7, arrowheads mark an ERMES complex moving into the growing bud). Several examples of ERMES puncta forming de novo were also documented ( Figure 7, open circles).
We used fluorescence recovery after photobleaching (FRAP) to determine whether Mmm1-GFP could be incorporated into an existing ERMES puncta. As shown in Figure 8, we detected recovery of Mmm1-GFP fluorescence 15 minutes after photobleaching (compare C and D). Additional fluorescence recovery was observed 30 minutes and 45 minutes after photobleaching ( Figure 8E and F). Thus, a mobile fraction of Mmm1-GFP is able to repopulate ERMES after photobleaching. No recovery of Mmm1-GFP was observed with time periods shorter than 15 minutes, indicating that the rate of recovery is   extremely slow and that ERMES structures are generally long lived. Together, these results are consistent with the idea that stable ERMES complexes have a structural role at ER-mitochondrial contact sites.

Discussion
In this report, we provide evidence that the ERMES complex and Gem1 do not directly affect lipid transport (6,24) or mitochondrial inheritance (7,8,11,12,23) as previously suggested. In addition, ERMES and Gem1 are likely functioning in distinct pathways, despite their genetic linkage and physical association (6,24,25). Our results are more consistent with a model in which the ERMES complex serves a structural role in maintaining the morphological integrity of mitochondria.
We found that the absence of ERMES components has little or no effect on the conversion of PS to PE. Thus, there is no dramatic defect in the transport of PS from the ER into mitochondria, and the small reduction in PS to PE conversion can be explained by an indirect effect of ERMES components on the import of Psd1 into mitochondria. The absence of Gem1 had no significant effect on PS to PE conversion. Consistent with our conclusions, the cellular phospholipid composition changed only slightly in mutants lacking both Psd2 and either ERMES components or Gem1. Kornmann et al. (6) did not actually report on PS to PE conversion, but they found a moderate decrease in the conversion of PS to PC in ERMES mutants. In our experiments, ERMES mutations did not affect the incorporation of radioactivity into PC ( Figure S3A). In addition, minor radiolabeling of PC can occur by other pathways (i.e. the 1-carbon pathway), making it difficult to reach conclusions based on analysis of PC alone. It should also be noted that we were unable to restore tubular mitochondrial morphology in gem1 and ERMES mutants by adding t = 0 minutes t = 20 minutes t = 40 minutes lipid precursors, including ethanolamine, choline, lyso-PE or lyso-PC ( Figure S5). Thus, mitochondrial morphology defects observed in gem1 and ERMES mutants are not simply a consequence of a deficiency of PC and PE.
The ERMES proteins Mmm1, Mdm10 and Mdm12 have been proposed to attach mitochondria to the actin cytoskeleton for polarized movement of mitochondria during cell division (8,13). Indeed, there are numerous reports in the literature that ERMES mutants produce large buds lacking mitochondria (7,9,11,12). Here, we provide evidence that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology changes. First, we showed that abnormally shaped mitochondria in ERMES mutants are transported into the bud upon overexpression of the inheritancepromoting factor Ypt11. Thus, aberrant mitochondria are able to bind and move on actin filaments in the absence of ERMES components. Second, we observed that restoration of tubular mitochondrial morphology by the artificial tether ChiMERA is correlated with the rescue of mitochondrial inheritance defects in mdm12 cells. The ChiMERA protein is able to restore ER-mitochondrial contact sites and tubular mitochondrial morphology but has no known actin-binding activity. Thus, mitochondrial shape is a more important determinant of mitochondrial distribution than the presence or absence of ERMES. Our combined findings argue against a model in which ERMES has a core function in mitochondrial association with, or movement along, the actin cytoskeleton.
ChiMERA expression was not able to rescue mitochondrial morphology defects in cells lacking Gem1 or the integral membrane proteins Mmm1 or Mdm10. Thus, Mdm10, Mmm1 and Gem1 carry out critical functions affecting mitochondrial shape, which cannot be bypassed by artificially tethering ER and mitochondria. Unambiguous determination of the primary molecular functions of Mdm10, Mmm1 and Gem1 in budding yeast is required to understand their roles in mitochondrial morphology maintenance.
Two groups have identified the Gem1 GTPase as a sub-stoichiometric component of the ERMES complex (24,25). We have not been able to co-precipitate untagged Gem1 with ERMES components, although we can detect untagged Gem1 in whole cell extracts (unpublished observations). Based on these results, it seems likely that only a small fraction of cellular Gem1 associates with assembled ERMES complexes. Consistent with this hypothesis, our fluorescence microscopy and immunoprecipitation experiments indicate that ERMES complexes persist when Gem1 is absent. Despite a recent report that loss of Gem1 decreases the number and increases the size of ERMES puncta in vivo (24), we did not observe significant differences in the number and size of ERMES puncta in WT and gem1 strains ( Figure 5 and unpublished observations). Thus, Gem1 does not appear to regulate the assembly or maintenance of ERMES complexes. Since Gem1 contains GTPase domains and calcium binding motifs (30), it probably has a regulatory role, but its function remains to be elucidated.
Using time-lapse imaging, we showed that many ERMES puncta represent stationary entities. However, we also found mobile puncta, suggesting the existence of behaviorally distinct classes of ERMES structures. FRAP experiments demonstrated that the exchange of Mmm1-GFP into preexisting ERMES puncta is slow, indicating that many ERMES structures are long-lived. Some of the ERMES puncta photobleached rapidly and could not be imaged, and might represent a class of less stable, or structurally distinct, ERMES complexes.
However, it appears that many ERMES complexes are simply static tethers between the ER and mitochondria.
Our results are most consistent with a model in which the ERMES complex serves as a physical link between ER and mitochondria, which is required for the maintenance of the normal morphology of mitochondria. In this model, ER structure would influence mitochondrial morphology through the ERMES complex. This is consistent with the observation that disruption of the ERMES complex leaves ER morphology intact, but disturbs the morphology of mitochondria. It is also supported by the recent observation that crossing points of ER tubules with mitochondria determine the sites of mitochondrial fission (31). The proposed model does not contradict the observation that individual ERMES components have additional functions, such as in the insertion of ß-barrel proteins into the outer membrane of mitochondria (15,16,32). On the other hand, the model suggests that some defects observed in ERMES mutants are simply a secondary consequence of mitochondrial morphology distortion.

Strains and plasmid construction
Standard methods were used for growth and analysis of yeast strains and plasmid construction (33). Yeast strains and plasmids are listed in Tables S1 and S2. p415-ADH was generated by PCR amplification of the ADHpr using forward primer. 5 -CCGgagctcGGTGTACAATATGGACTT and reverse primer 5 -GCtctagaTGTATATGAGATAGTTGATT that was cloned into p415 using the restriction sites SacI and XbaI. The PSD1 open reading frame was PCR amplified and cloned into p415-ADHpr, using restriction sites BamHI and XhoI.

Phosphatidylserine conversion assay (in vivo)
For measuring PS to PE conversion in vivo ( Figure 1A), overnight cultures were grown in SC dextrose medium containing 5 mM ethanolamine (Etn) and diluted in fresh media to resume growth. Aliquots of logarithmically grown cells (A600 of 0.3 OD) were sedimented by centrifugation and resuspended in 1 mL of fresh minimal medium, containing 5 mM Etn and 1 μCi L-[ 14 C(U)]-serine. Samples were incubated for 2 h at 30 • C followed by lipid extraction using the Folch procedure. In brief, cells were sedimented by centrifugation, resuspended in 330 μL of methanol and disrupted by vortex mixing with 100 μL of glass beads, for 10 min. Subsequently, 660 μL of chloroform was added and samples were centrifuged for 10 min at 10 000 x g to remove insoluble material. The organic solvent supernatant was recovered and washed once with 0.9% NaCl and the lower chloroform phase was dried under a stream of nitrogen. Lipids were resuspended in 15 μL chloroform and separated by TLC on Silica60 plates using chloroform/methanol/25% ammonium hydroxide (50/25/6 v/v/v). Labeled lipids were visualized using Phosphoimager (BioRad, PMI) and quantified using Metamorph software. Data reported are the mean ± SD for three experiments.
For measuring PS to PE conversion in vivo in Figure 2A, the psd2 and psd2 gem1 strains were grown in synthetic complete medium plus 2 mM Etn with glucose as a carbon source (SC). Cells in mid-log phase were harvested by centrifugation and washed twice by resuspension in water and recentrifugation. The cells were suspended in SC medium, at an A600 of 0.35 in a volume of 2 mL. Radiolabeling was initiated by adding 20 μCi L-[ 3 H(G)] serine, and growth was continued at 30 • C for 2 h with vigorous shaking. Labeled phospholipids were extracted as previously described (34) and the lipid classes were resolved by thin layer chromatography, and radioactivity was quantified by liquid scintillation spectrometry.

Phosphatidylserine conversion assay (in vitro)
For reconstituted aminoglycerophospholipid synthesis and transport ( Figure 1B), yeast cells were grown to early-logarithmic phase in synthetic dextrose media at 30 • C. Crude mitochondria (containing ER-mitochondrial contact sites) were purified as previously described (35). For lipid synthesis and transport, 100 μL crude mitochondria (100 μg protein) in 0.6 M mannitol, 20 mM Tris pH 7.4 and 0.6 mM MnCl 2 were incubated with 0.1 μCi L-[ 14 C(U)]-serine at 30 • C. After 20 min, 40 mM unlabeled serine was added and PS synthesis was arrested by addition of 5 mM EDTA. The conversion of nascent PS to PE was subsequently followed in a 45 min incubation at 30 • C. The reaction was stopped by adding 1 mL chloroform:methanol 2:1 (v/v). After shaking for 1 h, lipids were purified by extracting the organic phase, washing with 100 μL 0.9% NaCl (w/v) and drying at 65 • C. Lipids were resuspended in 15 μL chloroform, loaded on thin-layer chromatography plates (Silica 60), separated in chloroform/methanol/25% ammonium hydroxide (50/25/6 v/v/v) and visualized using a phosphoimager (BioRad, PMI). Radioactive lipids were quantified using ImageJ software. Data reported are the mean ± SD for three experiments.
For the aminoglycerophospholipid synthesis and transport studies with crude mitochondria shown in Figure 2B the process was followed using L-[3-3H(G)] serine incorporation into lipids as previously described (36). Briefly, 20 μCi of L-[3-3H(G)] serine (32 Ci/mmol) and 0.5 mM MnCl 2 were added to 100 μL crude mitochondria (100 μg protein) in buffer C (0.6 M sorbitol, 20 mM HEPES-KOH pH 7.4) and incubated at 30 • C for 15 min followed by incubation with EDTA (4 mM) for an additional 75 min. The initial incubation radiolabels the PS pool in the ER, and the subsequent incubation with EDTA arrests PS synthesis and allows for transport of the nascent PS to mitochondria for conversion to PE by Psd1. Radiolabeled aminoglycerophospholipids were isolated by lipid extraction and separated by thin layer chromatography on silica Gel H plates, using the solvents chloroform, methanol, 2-propanol, 0.25% KCl, triethylamine (30:9:25:6:18, v/v). The resolved radioactive lipids were quantified by liquid scintillation spectrometry (34). Data reported are the mean ± SD for three experiments.

Lipidome analysis by mass spectrometry
Samples for lipidome analysis were harvested from 20 mL cultures of yeast growing exponentially in YP-rich medium containing 2% glucose, washed in water at 4 • C, and frozen immediately in liquid nitrogen. Lipid analysis was performed using a Triversa NanoMate ion source (Advion Biosciences, Inc.) and a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) as previously described (37). PC and PE species were monitored in positive ion mode, and PS and PI species were monitored in negative ion mode. Lipids were quantified by intensity profiling using peak intensities of monitored lipid species normalized to the total intensity of all monitored lipid species within a FT MS scan. Samples were analyzed in duplicate.

Coimmunoprecipitation assays
Membrane fractions were prepared from logarithmically grown cells (YPD, 500 OD 600 units For FRAP experiments, cells expressing Mmm1-GFP mounted in SD media containing 2% low melting point agar were visualized using a Zeiss LSM 510 ConfoCor 3 microscope with a 100×, 1.4 numerical aperture (NA) oil-immersion objective (optical sections = 0.9 μm). To obtain 100% loss of fluorescence, an Mmm1-GFP puncta defined in an oval-shaped region of interest in the equatorial plane of the cell membrane was bleached using a 488 nm laser. FRAP was monitored using low laser intensity at 15-minute intervals. Projection view images were assembled and analyzed using ImageJ, Adobe Photoshop and Adobe Illustrator CS5.1.
Morphology and inheritance of mitochondria labeled with mtGFP (for Ypt11 expression and lipid supplementation studies) or mtRFP (for ChiMERA expression studies) were scored in log phase strains as described previously (23). For Ypt11 overexpression experiments, strains containing p416-MET25-YPT11 or p416-MET25 and the mtGFP marker were grown overnight at 30 • C in SD dropout media containing 0.1 mg/mL methionine. Ypt11 expression was induced for 4 h by diluting into SD media lacking methionine and cysteine. Quantification was performed as described above.
Figure S1: Mitochondrial PE biosynthesis pathway. PA is converted to PS in the ER and is subsequently transferred to the mitochondrion. PS is decarboxylated by Psd1 to form PE. Mitochondrial PE can be transferred to the ER for PC synthesis.  Figure 1A and Figure S3). B) Yeast cells (BY4741) were pulsed (3 h) and chased (4 h) with radiolabeled serine. The graph shows the percentage of radioactivity in PS, PE and PC during the chase phase. Samples were analyzed in duplicate and average values are shown. The maximal difference between two duplicate measurements was 4%. Figure S3: Overexpression of Psd1 increases conversion of PS to PE in WT, ERMES-mutant, and psd1Δ psd2Δ strains. The small reduction in PS to PE conversion caused by the absence of mdm34 and mdm10 in Figure 1A might be indirect, since these ERMES components are involved in the insertion of ß-barrel proteins into the outer mitochondrial membrane, and these in turn are required for the import of proteins, such as Psd1, into the inner membrane. To test this possibility, we measured radiolabeled serine incorporation into PS (black), PE (gray) and PC (white) in the indicated strains plus/minus a second copy of the PSD1 gene expressed from the constitutive ADH promoter. (A) Conversion of PS to PE was increased in both WT and ERMES-mutants. (B) Compromised PE synthesis in a psd1 psd2 strain lacking phosphatidylserine decarboxylases is rescued by Psd1 overexpression. For reasons that are unclear, these results differ from previous reports that Psd1 is not rate-limiting for PS to PE conversion (41)(42)(43). Additional studies are required to resolve this issue.    Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.