The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphorylation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulummembrane. The Nem1–Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of theNem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54–56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7D mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo. Consistent with the role of the Nem1–Spo7 complex in activating the function of Pah1, we found that themutational effects of the Spo7 LLI sequence were on the Nem1–Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphorylation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7D mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo. Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.
The activities of Pah1 PA phosphatase (10,42) and Nem1 protein phosphatase (31,43) are both dependent on the haloacid dehalogenase (HAD)-like domain with the DXDX(T/V) catalytic motif (Fig. 2). For Pah1, in addition to the catalytic domain, its N-LIP domain is also important for PA phosphatase activity (42). Pah1 is a peripheral membrane protein that translocates to the ER membrane via its dephosphorylation by the Nem1-Spo7 complex. For Pah1 translocation, its acidic tail at the C terminus is required to interact with the Nem1-Spo7 phosphatase complex (33), whereas its amphipathic helix at the N terminus is crucial to associate with the membrane surface (32). The tryptophan residue of Pah1 in the conserved sequence WRDPLVDID is essential for its in vivo function but is not required for catalytic activity (44). Nem1 and Spo7, which form a protein phosphatase complex, are integral membrane proteins, both of which contain two transmembrane domains (31). The complex formation of Nem1 with Spo7 occurs through its C-terminal conserved domain (31). The interaction of Spo7 with Pah1 (38) facilitates the formation of an enzyme-substrate complex between Nem1 and Pah1 (33).
In the present work, we examined the structural requirement of Spo7 in regulating the function of the Nem1-Spo7/Pah1 phosphatase cascade. Deletion analysis of Spo7 indicated that its amino acid region 46-65 is required for Nem1-Spo7/Pah1 axis function, and the N-terminal region contains the hydrophobic sequence Leu-Leu-Ile (LLI) (residues 54-56), conserved in fungi. Mutational analysis showed that the overall hydrophobicity of the LLI sequence is required for the complex formation of Spo7 with Nem1, and its defect causes a significant loss of the phosphatase complex-controlled Pah1 function (e.g. TAG synthesis and lipid droplet formation, the nuclear/ER membrane morphology, vacuole fusion, and growth at elevated temperature and on glycerol as a carbon source).

Identification of Spo7 LLI sequence as a functional requirement
To assess the structural requirement of Spo7 for its function, we mutated the 59 coding region of SPO7 to produce the nested deletions of the protein from the N terminus (Table 1). The mutant alleles of SPO7 on a low-copy-number plasmid were transformed into the spo7D mutant and evaluated for their ability to complement the mutant phenotypes. Loss of Spo7, like that of Pah1 or Nem1, renders cells defective in growth at 37°C ( Fig. 3) (10,31,43,45). Accordingly, we first examined whether the N-terminally truncated forms of Spo7 complement the growth defect of the spo7D mutant (Fig. 3). Like the full-length Spo7, its truncation forms Spo7D  and Spo7D  complemented the spo7D temperature-sensitive phenotype. However, the expression of Spo7D(2-65) did not complement the mutant phenotype. These results indicate that the loss of Spo7 function is caused by the lack of its sequence consisting of amino acids 46 to 65. A protein Blast analysis of the Spo7 sequence revealed the amino acid stretches that are conserved in fungi. We focused on one stretch, namely, LLI (residues [54][55][56], with the notion that hydrophobic interactions (46) govern the formation of the Nem1-Spo7 phosphatase complex. To examine this possibility, we constructed the SPO7 allele whose product lacks only the LLI sequence. The expression of the LLI-deficient Spo7, Spo7D(54-56), did not complement the spo7D temperature-sensitive phenotype (Fig. 3), showing that the hydrophobic sequence is required for Spo7 function.
We next examined the importance of the hydrophobicity of the LLI sequence by mutating each individual amino acid to alanine and arginine. The alanine substitution conserves the hydrophobic property of the leucine and isoleucine residues, whereas the arginine substitution introduces the hydrophilic property instead. The expression of the alanine-substituted forms (i.e. L54A, L55A, and I54A) of Spo7 complemented the spo7D temperature-sensitive phenotype (Fig. 3). In contrast, the argininesubstituted forms did not complement (L54R and L55R) or weakly complemented (I56R) the spo7D temperature sensitivity (Fig. 3). These results indicate that the hydrophobic property of the LLI sequence is important for Spo7 function, and that the hydrophobic requirement is more stringent for the leucine residues.

Spo7 LLI sequence interacts with Nem1
Spo7 functions as a regulatory subunit of the Nem1-Spo7 phosphatase complex (31,43). To address the hypothesis that the LLI sequence is required for the protein-protein interaction of Spo7 and Nem1, we examined the mutational effects of the hydrophobic sequence on Nem1-Spo7 complex formation. In this analysis, the isolation of the phosphatase complex and its immunodetection were facilitated by using the protein A-tagged Nem1 and the Myc-tagged Spo7 (20,31,34,40), which are functional to complement the spo7D phenotypes (e.g. temperature sensitivity and growth defect on glycerol medium).
The presence of Nem1-PtA in the isolated complex could be determined by immunoblot analysis with anti-protein A or anti-Nem1 antibody. However, anti-protein A antibody was more robust in signal detection; thus, it was used in this analysis. The cross-reactivity of anti-Spo7 antibody with proteins whose electrophoretic mobility overlaps that of untagged Spo7 interfered with the detection of the Spo7-specific signal. To circumvent this problem, we utilized the Myc-tagged version of Spo7 (31) that exhibits slower electrophoretic mobility for clear analysis with anti-Spo7 antibody. Although anti-Myc antibody detected Spo7-Myc, its signal was not as robust as that detected with anti-Spo7 antibody. Nem1 and Spo7 are very-low-abundance proteins (47), and their detection from cell extracts was The expression of PAH1 is regulated during growth by nutrient status as mediated by the Opi1/Ino2-Ino4 (Henry) regulatory circuit and transcription factors Gis1, Rph1, and Zap1 (48,109). Pah1 is phosphorylated (small white circles) by multiple protein kinases in the cytosol and translocates to the nuclear/ER membrane through its dephosphorylation by the Nem1-Spo7 complex (31,32). Dephosphorylated Pah1 that is associated with the nuclear/ER membrane via its amphipathic helix (32) catalyzes the conversion of PA to DAG (10), which is converted to TAG for storage in lipid droplets (LDs). The DAG is also used for the synthesis of phosphatidylcholine or phosphatidylethanolamine via the Kennedy pathway when cells are supplemented with choline or ethanolamine (7,110). Unphosphorylated/dephosphorylated Pah1 or protein kinase C (PKC)-phosphorylated Pah1 (16) is degraded by the 20S proteasome (indicated by the dashed-line arrows) (36).
untenable by immunoblotting with available antibodies. Thus, the typical input (i.e. cell extract) control was not applicable to the Nem1-Spo7 interaction data presented here.
Nem1-PtA was affinity purified with IgG-Sepharose from the extracts of NEM1-PtA spo7D cells expressing Spo7-Myc, and its level was determined by immunoblot analysis with antiprotein A antibody (Fig. 4A). In addition, Spo7-Myc copurified with Nem1-PtA was detected by immunoblot analysis with anti-Spo7 antibody (Fig. 4A). The quantification of Spo7-Myc showed that the amounts of the alanine-substituted forms (i.e. L54A, L55A, and I56A) were similar to that of the WT protein (Fig. 4B). However, the levels of the arginine-substituted forms L54R, L55R, and I56R were greatly reduced by 93, 85, and 63%, respectively. This result suggested that the hydrophobicity of the LLI sequence is crucial for Spo7 to form a complex with Nem1. However, we cannot rule out the possibility that the arginine mutations caused reduced stability of the Spo7 protein.
For Nem1-PtA, its cellular levels were shown to correlate with the levels of copurified Spo7-Myc (Fig. 4B). Unlike the alanine-substituted forms (i.e. L54A, L55A, and I56A) of Spo7, its arginine-substituted forms, L54R, L55R, and I56R, had the effect of reducing the Nem1-PtA level by 78%, 80%, and 44%, respectively. Considering that the Nem1-PtA level is reduced by 85% in the cell lacking Spo7-Myc expression (i.e. vector control), this result raises the suggestion that Nem1 is unstable when it cannot form a complex with Spo7.

Spo7 LLI sequence is required for the Nem1 catalytic activity
The Nem1-Spo7 phosphatase complex catalyzes the dephosphorylation of native Pah1, which is shown by a small increase in its electrophoretic mobility in SDS-PAGE (13,14,20). In contrast, the phosphorylation of Pah1 at Ser-723, Ser-744, and Ser-748 by the Pho85-Pho80 protein kinase is shown by a small decrease in its migration in the polyacrylamide gel (13,14,20). The Pho85-Pho80-mediated phosphorylation in the cell is required to ensure that the localization of Pah1 to the membrane depends on its dephosphorylation by the Nem1-Spo7 phosphatase (20). Accordingly, we examined the Nem1-Spo7 phosphatase activity in vivo on the substrate Pah1 using its phosphorylation state, which is reflected by differential electrophoretic mobility (Fig. 5). Compared with the expression of Spo7 (i.e. WT), the lack of its expression (i.e. vector) resulted in the production of the slower-migrating form of Pah1 concomitant with the lack of its faster-migrating form, indicating that the Nem1-Spo7 phosphatase activity is required to dephosphorylate native Pah1.
The differential electrophoretic mobility of Pah1 was also shown from spo7D cells expressing the alanine-and argininesubstituted forms of Spo7 (Fig. 5). The faster-migrating form of Pah1 was shown from the expression of the L54A form, similar to the expression of WT Spo7. In contrast, the slower-migrating form of Pah1 was shown from the expression of the L54R form, similar to the lack of WT Spo7. Compared with the L54A form of Spo7, the L55A and I56A forms showed a less clear effect on producing the faster-migrating form of Pah1. However, the electrophoretic mobilities of Pah1 from the expression of L55A and I56A were shown to be faster than those from the expression of L55R and I56R. The L55R and I56R forms were similar to the L54R form in producing the slower-migrating form of Pah1.

Effect on lipid content
Spo7 is required for the Nem1 catalytic activity, which in turn regulates the Pah1 function to produce DAG for TAG synthesis (10, 20, 32-34, 43, 48). Accordingly, we examined the effect of the site-specific mutations of the LLI sequence on lipid contents (Fig. 6). Lipids were analyzed from the [2-14 C]acetatelabeled cells at the stationary phase of growth, when Pah1 PA phosphatase activity is highest (10,48,50). Lack of Spo7 (i.e. vector control) caused a 92% decrease in the amount of TAG but a 96% increase in the amount of phospholipids. The altered lipid levels of spo7D cells were not restored by expressing the arginine-substituted forms (i.e. L54R, L55R, and I56R) of Spo7. However, the expression of the alanine-substituted forms L54A and L55A restored the altered lipid levels caused by the spo7D mutation. Interestingly, the I56A form of Spo7, which complemented the spo7D temperature-sensitive phenotype, showed a modest increase in the TAG level without restoring the phospholipid level. The differential effects shown by the I56A form supports the notion that a lower level of Pah1 PAP activity is sufficient to support cell growth at elevated temperature (44).

Effect on lipid droplet formation
Pah1 produces DAG that is acylated to TAG (10), which is then stored in lipid droplets (49). Since the Pah1-controlled production of TAG affects lipid droplet formation, we examined the mutational effects of the LLI sequence on the quantity of cellular lipid droplets (Fig. 7B). The spo7D cells expressing WT Spo7 contained an average of 9 lipid droplets in the stationary phase of growth, whereas the mutant cells lacking its expression showed a reduction of the lipid droplet number of 62%. Compared with the effect of the WT control, the argininesubstituted forms (i.e. L54R, L55R, and L56R) of Spo7 did not significantly increase the lipid droplet number of the spo7D cells. In contrast, the expression of the alanine-substituted forms L54A and L55A restored the lipid droplet number to 80% of the WT control. Similar to its effect on the TAG level,

Effect on nuclear/ER morphology
The lack of Spo7, Nem1, or Pah1 in the cell is characterized by an irregularly shaped nucleus with the expansion of the nuclear/ER membrane (Fig. 8A) (31,42,43). This phenotype is attributed to the increased synthesis of membrane phospholipids as caused by the defect of the Nem1-Spo7/Pah1 phosphatase cascade (4,9,10,43,48,54). Accordingly, we examined the mutational effects of the LLI sequence on the nuclear/ER morphology of the cell by the expression of the ER marker Sec63-GFP (Fig. 8B). On average, about 60% of spo7D cells exhibited aberrant nuclear/ER morphology compared with only 3.5% of WT cells. The expression of the alanine-substituted forms of Spo7 significantly reduced the aberrant nuclear/ER morphology of spo7D cells (14-22%), whereas the expression of the arginine-substituted forms had weaker effects on suppressing the mutant phenotype (37-42%).

Effect on vacuole fusion
A phenotype characteristic of the cell lacking Pah1, Nem1, or Spo7 is the appearance of fragmented vacuoles that fail to fuse upon nutrient limitation (Fig. 9A) (51, 53). Thus, we examined the effects of the site-specific mutations of the LLI sequence on the vacuole morphology of the cell by staining with FM 4-64. The defect of vacuole fusion was less pronounced in the spo7D mutant than in the pah1D or nem1D mutant (Fig. 9A, lower). As shown in Fig. 9B, about 18% of spo7D cells possessed fragmented vacuoles compared with 2.5% of the mutant cells expressing WT Spo7. The expression of the alanine-substituted forms of Spo7 restored the defective vacuole fusion of spo7D cells to a level similar to that of the cells expressing the WT protein. In contrast, the expression of the arginine-substituted forms had little effect on restoring the defective vacuole fusion of spo7D cells (L54R, 15%; L55R, 15%; I56R, 12%).

Effect on growth with glycerol as the carbon source
Cells lacking Pah1 or Nem1 exhibited a growth defect when glycerol was substituted for glucose as a sole carbon source (Fig. 10A) (42,45,55). Because the Nem1-Spo7 complex regulates Pah1 function, we examined whether spo7D mutant cells utilize glycerol as a carbon source. The spo7D cells exhibited a subtle loss-of-growth phenotype when grown on the glycerolcontaining medium; this phenotype was complemented by the expression of WT Spo7 (Fig. 10B). The alanine-substituted forms (i.e. L54A, L55A, and I56A) of Spo7 also complemented the growth defect of spo7D cells on glycerol medium. However, the arginine-substituted forms (i.e. L54R, L55R, and I56R) did not complement the spo7D defect in glycerol utilization.

Discussion
SPO7, which was originally identified as a gene involved in meiosis and sporulation (56), was shown to encode a protein required to maintain a spherical nuclear morphology (31). The role of Spo7 in sporulation is unclear, but its role in the nuclear morphology is based on the formation of a protein phosphatase complex with Nem1 to regulate Pah1 PA phosphatase (20,43). The dephosphorylation of Pah1 by the Nem1-Spo7 phosphatase at the nuclear/ER membrane (20,32,33) facilitates its membrane localization for catalytic activity on PA to produce DAG that is acylated to TAG (Fig. 1) (48). The storage lipid TAG is a reservoir for energy production as well as for growth resumption from stasis, and its synthesis protects cells against fatty acid-induced toxicity (50,(57)(58)(59)(60)(61)(62)(63). TAG production also has the effect of controlling the level of PA that is used for the de novo synthesis of membrane phospholipids. Accordingly, the disruption of the Nem1-Spo7/Pah1 phosphatase cascade by Spo7 deficiency causes the accumulation of PA that is exclusively converted to membrane phospholipids. The elevated level of PA also upregulates the production of phospholipid synthesis enzymes at the transcriptional level through the derepression of enzyme gene expression (10,43,48,50,54,64). These changes lead to a massive increase in the level of phospholipids at the nuclear/ER membrane, causing the organelle membrane to be irregularly expanded. The lack of the Spo7mediated control of the Nem1-Spo7/Pah1 phosphatase cascade also causes defects in other cellular processes (e.g. lipid droplet formation, vacuole fusion, and growth at elevated temperature and on glycerol medium) that are dependent on the regulatory function of PA and/or DAG.
In the Nem1-Spo7 phosphatase complex, Spo7 is a regulatory subunit for the function of the Nem1 catalytic subunit (31,34,38,43,65) and associates with its HAD-like domain (i.e. catalytic domain) at the C-terminal region (Fig. 2) (31). The work presented here is consistent with the notion that the LLI sequence of Spo7 at the N-terminal region is required for complex formation, and that the overall hydrophobicity of the amino acid residues is crucial for the protein-protein interaction with Nem1. It has been known that residue hydrophobicity is a dominant factor for protein-protein interactions (66). Thus, our findings indicate that the LLI-mediated hydrophobic interaction is a key factor for the formation of the Nem1-Spo7 complex. This mechanism of complex formation could be further supported by the identification of hydrophobic residues in the Nem1 catalytic domain that directly interact with Spo7 LLI. Obviously, the interaction of Spo7 with Nem1 would be better understood from the molecular structural information of the protein complexes. Our results also showed that Nem1 is unstable without forming a complex, indicating that Spo7 is required not only for the function of the catalytic subunit but also for its protein stability. The effect of complex formation on the stability of the catalytic subunit is conserved, as shown by the instability of human CTDNEP1 (catalytic subunit, also called Dullard) in the absence of complex formation with NEP1-R1 (regulatory subunit, also called TMEM188) (65,67).
The orthologous components of the yeast Nem1-Spo7/Pah1 phosphatase cascade in higher eukaryotes consist of the CTDNEP1-NEP1-R1 complex (65,67) and lipin PA phosphatase (10,(68)(69)(70). Like yeast Pah1, mammalian lipin is controlled for its localization and PA phosphatase activity (71-74) by phosphorylation (75-81) and dephosphorylation (65, 67, 82).  Spo7 sequence LLI is required for Nem1-Spo7/Pah1 function In addition, the loss of lipin PA phosphatase is responsible for a variety of lipid-based metabolic disorders. Lipin 1 deficiency in mice and humans causes rhabdomyolysis (83,84), and its deficiency in mice is also characterized by hepatic steatosis during the neonatal period, lipodystrophy, insulin resistance, and peripheral neuropathy (68,85). The polymorphism of human LPIN1 is associated with insulin resistance and the metabolic syndrome (86). The deficiency of lipin 2 in humans results in chronic recurrent multifocal osteomyelitis and congenital dys-erythropoietic anemia (87,88), whereas the specific polymorphisms of human LPIN2 are associated with type 2 diabetes (89). In mouse small intestine, lipin 2 and lipin 3 deficiencies disrupt phospholipid homeostasis, impairing lipoprotein biogenesis and secretion (90). In mice, the loss of CTDNEP1 causes hemorrhagic ovarian cysts with the accumulation of red blood cells in follicles, leading to infertility (91). Unlike CTDNEP1, NEP1-R1 is not yet known for the effects of its deficiency. Nonetheless, the conservation of the phosphatase  cascade indicates that an important avenue of inquiry is to understand how NEP1-R1 interacts with CTDNEP1 for the regulation of lipin activity.
Spo7 is conserved in eukaryotes from fungi to humans (65) by containing two transmembrane-spanning domains (TM1 and TM2) and three homology regions (CR1, CR2, and CR3) (Fig. 2). Whereas the LLI sequence contained within CR1 is conserved in fungi, the two amino acid residues following the first conserved leucine vary in higher eukaryotes (65). Moreover, the second amino acid in the sequence of the worm Caenorhabditis elegans (LKF), the fly Drosophila melanogaster (LKA), and the human Homosapiens (LKA) is the charged amino acid lysine. Thus, based on the data from yeast Spo7, its orthologs containing the lysine residue following the conserved leucine residue are likely to have a weak interaction with the Nem1 orthologs in those organisms.
Compared with CR1 located at the N-terminal region, CR2 and CR3 are located at the C-terminal region. The N-terminal CR1 and the C-terminal CR2/CR3, which are outside the transmembrane regions, are both oriented to the cytosol. The requirement of Spo7 as a regulatory subunit for the catalytic function of Nem1 is thought to be in the recruitment of the substrate. This function of Spo7 requires a region interacting with Pah1, which is separate from a region required to form a complex with Nem1. The LLI sequence of Spo7 located in the N-terminal CR1 is required to form a complex with Nem1. Thus, it is possible that CR2 and CR3 are involved in the recognition of Pah1. In this regard, work is currently in progress to assess the role of CR2 and CR3 in the interaction with Pah1.

Experimental procedures Materials
Avanti Polar Lipids was the source of lipid standards. Bio-Rad supplied the molecular mass protein standards and  A, WT, pah1D, nem1D, and spo7D mutant cells were grown at 30°C in YPD to saturation, serially diluted (10-fold), and spotted onto agar plates containing 2% glucose or 3% glycerol. B, spo7D (GHY67) transformants expressing the indicated SPO7 allele (pGH443 and its derivatives) were grown in SC-Leu medium and treated as in panel A. Growth on glucose and glycerol was scored after 3 and 6 days, respectively, of incubation at 30°C. The data are representative of three independent experiments. Spo7 sequence LLI is required for Nem1-Spo7/Pah1 function reagents for electrophoresis, immunoblotting, and protein determination. DNA size standards were from Invitrogen. Carrier DNA for yeast transformation was from Clontech. Growth media were from Difco Laboratories. GE Healthcare was the source of IgG-Sepharose, polyvinylidene difluoride (PVDF) membrane, and the enhanced chemifluorescence Western blotting detection kit. FM 4-64 was purchased from Molecular Probes, Inc. Millipore-Sigma was the supplier of ampicillin, BSA, 2-mercaptoethanol, PCR primers, nucleotides, Ponceau S stain, Triton X-100, protease and phosphatase inhibitors, rabbit anti-protein A antibody (product P3775, lot 025K4777), rabbit anti-myc antibody (product no. SAB4301136, lot no. 802535536), and silica gel 60 TLC plates. Enzyme reagents for DNA manipulations and the Q5 site-directed mutagenesis kit were obtained from New England Biolabs. Radiochemicals were from Perkin-Elmer Life Sciences, and scintillation-counting supplies were from National Diagnostics. Qiagen was the source of plasmid DNA purification kits. Thermo Scientific was the source of alkaline phosphataseconjugated goat anti-rabbit IgG antibody (product 31340, lot NJ178812) and BODIPY 493/503. Anti-Nem1 (40), anti-Spo7 (40), and anti-Pah1 (13) antibodies were previously generated in New Zealand White rabbits. All other chemicals were reagent grade.

Strains, plasmids, and DNA manipulations
The strains and plasmids used in this work are listed in Table  1. Escherichia coli strain DH5a was used for plasmid maintenance and amplification. S. cerevisiae strains GHY67 (40) and GHY68 (40), which are spo7D mutants with chromosomal NEM1 and NEM1-PtA, respectively, were used for the expression of plasmid-borne SPO7 and its mutant derivatives. Plasmid pGH443 (40), which is a derivative of the E. coli/yeast shuttle vector pRS415 (92), directs the low-copy-number expression of SPO7 in S. cerevisiae. Plasmid pGH447 was derived from pGH443 by introducing a BamHI site before the stop codon of SPO7. pGH448 was constructed from pGH447 by inserting a 126-bp BamHI fragment for the 33Myc tag that was released from plasmid pRS314-SPO7-Myc (31). The derivatives of plasmids pGH443 and pGH448 with SPO7 mutations were constructed by the Q5 site-directed mutagenesis kit with appropriate primers designed using the NEBaseChanger online software. pGH449 was constructed by ligation of the SmaI/SacIdigested pRS413 (92) with the SEC63-GFP DNA that was released from YCplac33-SEC63-GFP (93) by digestion with Hin-dIII, treatment with Klenow fragment, and digestion with SacI. Standard methods were used for the isolation of chromosomal and plasmid DNA, for the digestion and ligation of DNA, and for the PCR amplification of DNA (94)(95)(96). Plasmid transformations of E. coli (95) and yeast (97) were performed as described previously. All gene mutations were confirmed by DNA sequencing.

Growth conditions
Bacterial cells were grown at 37°C in lysogeny broth medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4). E. coli transformants containing plasmids were selected by antibiotic (e.g. 100 mg/ml ampicillin) resistance. Standard methods were used for culturing yeast (94,95). Yeast cells were routinely grown at 30°C in rich or synthetic complete medium, and those containing plasmids were maintained in synthetic dropout medium. Unless otherwise indicated, 2% glucose was included as a carbon source in growth medium. Solid media for the growth of E. coli and yeast contained agar at a concentration of 1.5 and 2%, respectively. For the measurement of growth on solid medium, serially diluted (10-fold) cultures were spotted onto agar medium, and cell growth was scored after incubation for 3 days at 30 or 37°C (for temperature sensitivity). Growth on agar medium containing glycerol as the carbon source was scored after incubation for 6 days. Cell density in liquid cultures was estimated spectrophotometrically by measuring absorbance at 600 nm.

Preparation of cell extracts
All steps to prepare cell extracts were performed at 4°C. Yeast cultures were harvested by centrifugation at 1,500 3 g for 5 min. The cells were washed with water and resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 0.3 M sucrose, 10 mM 2mercaptoethanol) containing protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 mg/ml aprotinin, 5 mg/ml leupeptin, and 5 mg/ml pepstatin) and phosphatase inhibitors (2 mM imidazole, 1 mM b-glycerophosphate, 1 mM sodium pyrophosphate, 1 mM sodium fluoride, and 1 mM sodium orthovanadate). The suspended cells were mixed with glass beads (0.5-mm diameter) and then disrupted by five repeats of a 1-min burst and 2-min cooling using a BioSpec Products Mini-Beadbeater-16 (98). The disrupted cells were centrifuged at 1,500 3 g for 10 min to separate cell extracts (supernatant) from unbroken cells and cell debris (pellet). The protein concentration of the cell extracts was determined by the method of Bradford (99) using BSA as a standard.

Immunoblotting
Protein transfer from SDS-polyacrylamide gels to PVDF membranes was monitored by Ponceau S staining. The membrane blots were cut; the upper and lower portions were probed with rabbit anti-Nem1 (1 mg/ml) or rabbit anti-protein A (2 mg/ ml) antibodies and with rabbit anti-Spo7 (1 mg/ml) or rabbit anti-Myc (1:1,000 dilution) antibodies, respectively. The immunoblot analysis of Pah1 from cell extracts subjected to SDS-PAGE using 8% polyacrylamide gels was performed with rabbit Spo7 sequence LLI is required for Nem1-Spo7/Pah1 function anti-Pah1 antibody (2 mg/ml). The secondary goat anti-rabbit IgG antibody conjugated with alkaline phosphatase was used at a dilution of 1:5,000. Immune complexes on the PVDF membrane were detected using the enhanced chemifluorescence immunoblotting substrate. Fluorimaging, using a Storm 865 Molecular Imager (GE Healthcare), was used to acquire fluorescence signals from immunoblots, and the intensities of the images were analyzed by ImageQuant TL software (GE Healthcare). A standard curve was used to ensure that the immunoblot signals were in the linear range of detection.

Radiolabeling and analysis of lipids
The labeling of cellular lipids with [2-14 C]acetate was performed as described previously (105). Lipids were extracted (106) from the radiolabeled cells and then separated by onedimensional TLC in the solvent system of hexane-diethyl ether-acetic acid (40:10:1, v/v) (107). The resolved lipids were visualized by phosphorimaging and quantified by ImageQuant TL software. The identity of radiolabeled lipids was confirmed by comparison with the migration of authentic standards visualized by staining with iodine vapor.

Fluorescence microscopy
For the fluorescent staining of lipid droplets, yeast cells were grown at 30°C in synthetic medium to the stationary phase, incubated for 30 min with 2 mM BODIPY 493/503, washed with PBS (pH 7.4), and resuspended in the same buffer. For the fluorescent labeling of nuclear/ER membrane, yeast cells transformed with the SEC63-GFP plasmid (43) were grown at 30°C in the selection medium to the logarithmic phase and subjected to microscopic analysis. For the fluorescent staining of vacuole membranes, cells were grown at 30°C in synthetic medium to the late-logarithmic phase, incubated for 15 min at 30°C with 32 mM FM 4-64, washed with yeast extract peptone dextrose, and resuspended in fresh growth medium (108). The number of lipid droplets per cell and the percentage of cells with aberrant nuclear/ER morphology (misshaped versus round nuclei) or defective vacuole fusion (e.g. fragmented vacuoles) were scored from 4 fields of view (200 cells). The green and red fluorescence signals were examined under a Nikon Eclipse Ni-U microscope with the EGFP/FITC/ Cy2/AlexaFluor 488 and TRITC/Cy3/TagRFP/AlexaFluor 546 filters, respectively, re-corded by the DS-Qi2 camera, and subjected to imaging analysis with NIS-Elements BR software.

Analysis of data
Microsoft Excel software was used for the statistical analysis of data. p values of ,0.05 were taken as significant differences.

Data availability
All data are contained within the manuscript.