Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast

Lipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of ∼4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs; we identify 17 fld (few LDs) and 116 mld (many LDs) mutants. One of the fld mutants (fld1) is caused by the deletion of YLR404W, a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Δ cells demonstrate significantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fld1Δ cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Δ cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.


Introduction
Lipid droplets (LDs) are intracellular lipid inclusions consisting primarily of triacylglycerols (TAGs) and sterol esters (SEs; Murphy and Vance, 1999 ). Recent studies have proven that LDs are dynamic intracellular organelles that are functionally connected with other cellular compartments (for review see Martin and Parton, 2006 ). LDs from different cell types are bounded by a monolayer of phospholipids and characterized by unique surface proteins, which may contribute to the biogenesis, maturation, and stability of LDs ( Martin and Parton 2006 ;Wolins et al., 2006 ). LDs are believed to originate from microdomains of the ER, where enzymes for the synthesis of neutral lipids reside. Newly synthesized neutral lipids are thought to accumulate between two leafl ets of the ER bilayer before budding into the cytosol ( Martin and Parton 2006 ;Wolins et al., 2006 ). With the assistance of microtubules, nascent LDs appear to fuse with each other until a certain size is reached ( Bostrom et al., 2005 ). A recent study identifi ed SNARE proteins as part of the core machinery for the fusion of LDs ( Bostrom et al., 2007 ). Despite recent progress, many fundamental questions remain. For instance, how is the fusion process regulated, and what factors determine the fi nal size of a mature LD?

Results and discussion
In an effort to identify novel gene products that may play a role in LD formation, Nile red, a vital dye specifi c for intracellular LDs, was used to visually screen the entire collection of viable single-gene deletion mutants of the budding yeast Saccharomyces cerevisiae for abnormalities in the number and morphology of LDs ( Greenspan et al., 1985 ). Because the number and morphology of LDs may vary depending on growth phases, wildtype cells and all mutants were grown overnight to stationary phase (OD 600 = ‫ف‬ 5) in this study immediately followed by Nile red staining and fl uorescence microcopy. Wild-type cells at stationary phase showed 5.16 ± 2.18 LDs per cell on average ( ± SD; n = 200), and ‫ف‬ 80% of the cells displayed three to seven LDs ( Fig. 1 A , a). To simplify the screening process, we arbitrarily categorized deletion strains with the majority ( > 80%) of cells accumulating on average less than three LDs as fl d ( few LDs ) mutants and strains accumulating more than seven LDs as mld ( many LDs ) mutants. Among the mld mutants, strains containing > 11 LDs were classifi ed as strong mld mutants. We isolated L ipid droplets (LDs) are emerging cellular organelles that are of crucial importance in cell biology and human diseases. In this study, we present our screen of ‫ف‬ 4,700 Saccharomyces cerevisiae mutants for abnormalities in the number and morphology of LDs ; we identify 17 fl d ( few LDs ) and 116 mld ( many LDs ) mutants. One of the fl d mutants ( fl d1) is caused by the deletion of YLR404W , a previously uncharacterized open reading frame. Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1 ⌬ cells demonstrate sig nifi cantly enhanced fusion activities both in vivo and in vitro. Interestingly, the expression of human seipin, whose mutant forms are associated with Berardinelli-Seip congenital lipodystrophy and motoneuron disorders, rescues LD-associated defects in fl d1 ⌬ cells. Lipid profi ling reveals alterations in acyl chain compositions of major phospholipids in fl d1 ⌬ cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.
Fld1p, a functional homologue of human seipin, regulates the size of lipid droplets in yeast Figure 1. The fl d1 ⌬ (ylr404w ⌬ ) cells synthesize morphologically distinct LDs. (A) Both wild-type and fl d1 ⌬ cells were grown in YPD medium until stationary phase or until log phase. Cells were stained with 20 g/ml Nile red and immediately observed for LDs under a fl uorescence microscope. Micrographs of the wild-type cells (a and a Ј ) and fl d1 ⌬ cells (b -e, b ' , and c ' ) are shown. Supersized LDs (b, c, and b ' ) are indicated by arrows, and aggregation of LDs (c, d, and c ' ) is indicated by arrowheads. DIC, differential interference contrast. (B) Conventional TEM of wild-type and fl d1 ⌬ cells. Cells were grown in YPD to stationary phase, fi xed with 2.5% (vol/vol) glutaraldehyde and 2% (wt/vol) osmium tetroxide, and subjected to EM. LDs are seen as electron-transparent droplets. TEM of wild-type (a) and fl d1 ⌬ (b -i) cells. (C) Culture media affect LD morphology in fl d1 ⌬ cells. Wild-type and fl d1 ⌬ cells were grown until stationary phase in YPD medium (a and d), SC medium (b and e), YPO (oleate) medium (c and f), or YPDO medium (g). The preparation of media was described in Materials and methods. Nile red staining of wild-type (a -c) and fl d1 ⌬ (d -g) cells. Arrows indicate supersized LDs, and arrowheads indicate aggregated LDs. Bars: (A and C) 5 m; (B) 1 m. cultured in YPD media were only observed in about 10% of the cells grown in SC. Interestingly, when fl d1 ⌬ cells were cultured in YPO medium, > 95% (191/200 cells examined) of the cells displayed amorphous aggregations of LDs without the supersized LDs ( Fig. 1 C , f). However, when fl d1 ⌬ cells were cultured in YPDO medium (YPD + oleate), the large LDs appeared again together with the aggregation of smaller LDs ( Fig. 1 C , g). Thus, we identifi ed a commonly used growth medium (SC) that allowed us to easily distinguish wild-type and fl d1 ⌬ cells based on the presence or absence of supersized LDs ( Fig. 1 C , compare b with e).
We measured the steady-state levels of TAG and SE as well as the rate of oleate incorporation into TAG and SE ( Oelkers et al., 2002 ). For cells grown in YPD to log phase, the deletion of FLD1 caused about a doubling in the steady-state levels of both TAG and SE ( Fig. S1 A, available at http://www.jcb.org/ cgi/content/full/jcb.200711136/DC1). The rate of SE synthesis was also upregulated by 70% in fl d1 ⌬ deletion cells, but little difference in the rate of oleate incorporation into TAG was observed ( Fig. S1 B). Similar patterns of changes were detected for cells grown in YPD to stationary phase or in SC to either log or stationary phase (unpublished data).
The expression of FLD1-GFP in fl d1 ⌬ cells restored the normal morphology of LDs ( Fig. 2, A and B ). We performed a series of subcellular fractionation experiments to analyze the cellular distribution of Fld1p. Cell extracts prepared from the fl d1 ⌬ strain expressing Fld1-GFP were fractionated by centrifugation at 13,000 g for 10 min, resulting in P13 pellet and S13 supernatant fractions that were probed with antibodies against GFP and Dpm1p, an ER marker. Both Fld1p and Dpm1p were found in the P13 fraction, which contains large membranous structures such as the vacuole, ER, and plasma membrane ( Fig. 2 C ). The same cell extracts were subjected to continuous sucrose density gradient analysis. 13 fractions were collected from top to bottom (1 -13) and were probed for the presence of GFP and Dpm1p by immunoblotting. Dpm1p and Fld1p appeared to exist in the same density fractions ( Fig. 2 D ). Localization of Fld1-GFP was also examined in live cells by fl uorescent microscopy, and Fld1-GFP was found in both perinuclear and peripheral ER ( Fig. 2 E ). Finally, immuno-EM was used to pinpoint the exact location of Fld1p. Fld1p-GFP was found to be associated with the cortical ER and the nuclear envelope, which is consistent with the putative ER localization observed by light microscopy. In addition, labeling was observed throughout the ER, including regions in contact with LDs ( Fig. 2 F ).
The existence of morphologically distinct LDs within fl d1 ⌬ suggests enhanced fusion activities of LDs: the small, discrete LDs may represent the newly synthesized LDs, which tend to aggregate before eventually fusing into a supersized LD. To test this hypothesis, wild-type and fl d1 ⌬ cells were cultured in SC medium until midlog phase (OD 600 = ‫ف‬ 1.0), stained with Nile red, and observed for the fusion of LDs by fl uorescent microscopy. Cells in which two or several LDs lay close together were targeted. We examined 200 cases of adjacent LDs each for mutant and wildtype cells and monitored every case for 1 min. The criteria we used to defi ne fusion were described previously ( Bostrom et al., 2007 ). No fusion events were observed in wild-type cells, but 17 fl d mutants and 116 mld mutants (Tables S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1).
We focused on FLD1 , which corresponds to a previously uncharacterized ORF, YLR404W , because its deletion not only affected the number of LDs but also gave rise to strikingly enlarged or aggregated LDs ( Fig. 1 ). When grown in rich medium until stationary phase, wild-type cells usually display three to seven LDs under the microscope. The LDs were between 0.2 and 0.4 m in diameter and were almost spherical in shape ( Fig. 1 A , a). In contrast, LDs observed in fl d1 ⌬ cells were very irregular in terms of quantity, shape, and size. Up to 30% of the total population of fl d1 ⌬ cells contained one or a few supersized LDs that were spherical in shape and were about 0.5 -1.5 m in diameter ( Fig. 1 A , b; arrow), which means that the volume of the largest LD of the fl d1 ⌬ cells was about 50 times that of the largest LD found in wild-type cells. About 60% of the fl d1 ⌬ population contained an amorphous aggregation of neutral lipids in addition to several small LDs ( Fig. 1 A , c and d; arrowheads). The remaining ‫ف‬ 10% of the fl d1 ⌬ cells contained scattered and weakly stained LDs, which had diameters of < 0.1 m ( Fig. 1 A , e). The phenotypic characteristics of fl d1 ⌬ were also observed in log-phase cells. When grown to log phase (OD 600 = 0.8), most of the wild-type cells contained two or three LDs that were slightly smaller than those of stationary-phase cells ( Fig. 1 A , a Ј ). The LDs in fl d1 ⌬ at log phase show similar morphology to cells grown to stationary phase except that the supersized LDs and the aggregation of neutral lipids were smaller and more weakly stained ( Fig. 1 A , b Ј and c Ј ; arrow and arrowhead, respectively).
To examine the ultrastructure of fl d1 ⌬ cells, we performed transmission EM (TEM) of the wild-type and fl d1 ⌬ strains. Cells were grown in rich medium until stationary phase and were subjected to TEM analysis. One typical cross section of a wildtype cell contained fi ve LD profi les, which were round and about 0.2 -0.4 m in diameter ( Fig. 1 B , a). The cross sections of fl d1 ⌬ mutant cells again showed three classes of LDs with distinct morphologies. Up to 30% of cells displayed one or a few supersized LDs per section, which were either round or oval ( Fig. 1 B , b -d). Consistent with the result of fl uorescence microscopy, the diameters of some LDs were up to 1.5 m ( Fig. 1 B , d). Aggregated LDs were found in ‫ف‬ 60% of the mutant population ( Fig. 1 B , d -g). These aggregations were reminiscent of the amorphous neutral lipid clump observed under fl uorescence microscopy ( Fig. 1 A , c and d). The rest of the fl d1 ⌬ cells ( ‫ف‬ 10% of the total population) contained many tiny LDs, most of which had a diameter of < 0.1 m and were loosely scattered ( Fig. 1 B , h and i).
Yeast cells undergo marked proliferation of LDs when they are grown in defi ned medium (synthetic complete [SC]) or in an oleate-based medium (YPO [YP plus oleate, no glucose]; Binns et al., 2006 ). As shown in Fig. 1 C (a -c), a great increase in the number of LDs was observed in wild-type cells grown in SC or YPO media. When fl d1 ⌬ cells were grown in YPD, the majority ( ‫ف‬ 70%) of the cells showed amorphous aggregations of many intermediate-sized LDs ( Fig. 1 B , f and g). In contrast, > 70% of the fl d1 ⌬ cells displayed only one or two supersized LDs when cultured in SC media ( Fig. 1 C , e). Moreover, amorphous aggregation of many small LDs that were common in cells (C) Cells expressing Fld1-GFP were spheroplasted and subjected to differential centrifugation as described in Materials and methods. The 13,000-g pellet (P13) and soluble fraction (S13) were analyzed by SDS-PAGE and immunoblotting. (D) Cell lysates were loaded on the top of a continuous sucrose gradient (10 -53%) and centrifuged at 100,000 g for 15 h. Fractions were collected from the top, separated by SDS-PAGE, and immunoblotted with antisera against GFP or Dpm1. (E) Fluorescence microscopy and motorneuron disorders, was identifi ed ( Agarwal and Garg, 2004 ). Seipin shows weak sequence conservations to Fld1p, but they both contain two predicted transmembrane domains, and their predicted secondary structures are also very similar ( Fig. 4 A ). Human seipin is encoded by the BSCL2 gene, which gives rise to at least three different mRNAs and two peptides with 398 or 462 amino acids ( Lundin et al., 2006 ). The region that covers the first 280 amino acids of human seipin is 88% identical among rat, mouse, chimpanzee, and human homologues ( Agarwal and Garg, 2004 ). This is interesting because all of the sequence conservations between yeast Fld1p (285 amino acids) and seipin fall within this region. To test whether seipin is a functional homologue of Fld1p, full-length human and mouse seipin homologues were expressed in fl d1 ⌬ cells grown in SC media. The average diameter of the LDs is 1.27 ± 0.19 m ( n = 117) without human seipin and 0.43 ± 0.05 m ( n = 106) with seipin ( Fig. 4 B ). We also examined the effects of point mutations in seipin that are implicated in lipodystrophy (A212P) and motoneuron disorders (N88S and S90L). The expression of N88S and S90L but not A212P rescued the defects in LD morphology ( Fig. 4 C ).
fusion was detectable for about 10% (19/200) of all mutant cases. As shown in Fig. 3 A , two closely positioned LDs appeared to completely fuse within a span of 2 s. The size of the newly formed LD appeared to be the combined size of the two parent LDs.
To further demonstrate enhanced LD fusion in fl d1 ⌬ cells, we isolated LDs from wild-type and mutant cells both carrying Tgl3p-GFP. Tgl3p is a triglyceride lipase that localizes to the surface of LDs ( Fig. 3 B ; Athenstaedt and Daum, 2003 ). Purifi ed LDs from both wild-type and fl d1 ⌬ cells were left in PBS buffer and examined by microscopy before and after 60 min. Whereas LDs from wild-type cells remained scattered and unchanged in size, LDs from fl d1 ⌬ cells formed aggregates or fused into huge lipid inclusions reminiscent of the supersized LDs observed in live fl d1 ⌬ cells ( Fig. 3, C and D ). The lipid inclusions observed in vitro are much larger than the supersized LDs in live cells ( ‫ف‬ 5 m vs. ‫ف‬ 1 m in diameter), suggesting that additional fusion events occurred in vitro.
Remote homology detection techniques were used to look for a mammalian homologue to Fld1p. Human seipin, a protein associated with Berardinelli-Seip congenital lipodystrophy (BSCL) of cells expressing Fld1-GFP. DIC, differential interference contrast. (F) Localization of Fld1p by immuno-EM. Cells were grown in SC media to late log phase, fi xed, and processed for immuno-EM with antisera against GFP.  Congenital generalized lipodystrophy (CGL; or BSCL) is an autosomal recessive disorder that is characterized by the almost complete absence of adipose tissue and severe insulin resistance ( Agarwal and Garg, 2004 ). Genome-wide linkage analysis identifi ed two loci for CGL: CGL type 1 (CGL1) is caused by mutations in the AGPAT2 (1-acylglycerol-3-phosphate-O -acyl transferase 2) gene, and CGL2 is caused by mutations in BSCL2 , This is not surprising because A212P is considered a lossof-function mutation, whereas N88S and S90L may represent gain-of-function mutations ( Windpassinger et al., 2004 ). Lastly, expression of the highly conserved 280 -amino acid region of seipin rescued the defects in LD morphology ( Fig. 4 C ). Together, these results strongly suggest that human seipin represents the functional homologue of Fld1p. The alignment was created using the PRALINE server ( Simossis and Heringa, 2005 ). Identical amino acids among seipin homologues are shaded in black, and similar residues are shaded in gray. The second ary structures predicted by PSIPRED (positionspecifi c iterated predication of protein secondary structure) through the Ali2D server (http:// toolkit.tuebingen.mpg.de/ali2d) are indicated below the sequences. The boxed regions are the two transmembrane domains predicted through the PSIPRED server ( McGuffi n et al., 2000 ). (B and C) The expression of seipin in yeast. fl d1 ⌬ strains transformed with YC-plac111-MET3-BSCL2 -expressing full-length seipins (human BC012140 and AF052149 and mouse BC061689), human seipin (BC012140) amino acid residues 1 -280, seipin mutants, or the vector alone were grown in SC media minus leucine to stationary phase followed by Nile red staining and fl uorescence microscopy. Bars, 5 m. and lipodystrophy. Because mutations in AGPAT2 and BSCL2 cause similar clinical manifestations, we wondered whether aberrant phospholipid metabolism may underlie the cellular defects for both conditions. Lipid species from wild-type and fl d1 ⌬ whole cell extracts were analyzed by electrospray ionization tandem mass spectrometry. The level of PA increased slightly in fl d1 ⌬ cells (unpublished data). Interestingly, there is a shift from long-chain (18:1) to medium/short-chain (16:0, 14:0, and 12:0) which encodes seipin ( Magre et al., 2001 ;Agarwal et al., 2002 ). AGPAT2 catalyzes the formation of phosphatidic acid (PA), but knocking down AGPAT2 led to elevated levels of several phospholipid species, including PA, and to a delay in the activation of key transcription factors for adipogenesis such as C/EBP ␤ and PPAR ␥ ( Gale et al., 2006 ). Therefore, AGPAT2 controls adipogenesis through modulation of the synthesis of phospholipids. In contrast, little is known about the role of seipin in adipogenesis

Fluorescence microscopy
Fluorescence imaging was performed on a microscope (DMLB; Leica) with a fl uorescent lamp (ebq 100; Curtis). Samples were viewed using a 100 × NA 1.30 oil immersion objective lens (Leica). Images were taken with a digital camera (DFC480; Leica) and FW4000 software (Leica).

LD staining
Nile red (Sigma-Aldrich) is a specifi c and excellent vital dye for intracellular LDs. A 450 -490-nm bandpass excitation fi lter, a 510-nm dichromatic mirror, and a 515-nm longpass emission fi lter (fi lter cube I3; Leica) were chosen to view LDs.

Visualization of LD fusion
To record the process of LD fusion in the fl d1 ⌬ mutant, 3 l of late log-phase cells (OD 600 = ‫ف‬ 1) stained with Nile red were spotted on a slide and covered with a coverslip. Under the microscope, cells in which two or several LDs lay close together were targeted. Images were collected at 2-s intervals.

DAPI staining of nucleus
Cells fi xed with 3.7% formaldehyde for 15 min were stained with 5 g/ml DAPI. DAPI fl uorescence was observed with a 340 -380-nm bandpass excitation fi lter, a 400-nm dichromatic mirror, and a 425-nm longpass emission fi lter (fi lter cube A; Leica).

TEM
Cells were grown in rich medium until stationary phase, harvested, fi xed with 2.5% glutaraldehyde, and postfi xed with 2% (wt/vol) osmium tetroxide. The samples were subsequently dehydrated in a series of graded ethanol and embedded in Spurr ' s resin. 80-nm ultrathin sections were stained with uranyl acetate and lead citrate and examined under an electron microscope (JEM-1230; JEOL).

Immuno-EM
Immuno-EM of ultrathin cryosections was performed essentially as described previously ( Martin et al., 2005 ). In brief, fl d1 ⌬ cells transformed with FLD1-GFP were grown in SC media to late log phase and fi xed in 2% PFA/0.2% glutaraldehyde in 0.1 M PHEM buffer (60 mM Pipes, 25 mM Hepes, 2 mM MgCl 2 , and 10 mM EGTA), pH 6.9, for 1 h at room temperature. Cells were embedded in 10% gelatin, cryoprotected using polyvinyl pyrrolidone -sucrose, and snap frozen onto specimen holders in liquid N 2 . Ultracryomicrotomy was performed by a slight modifi cation of the Tokuyasu technique, and sections were picked up with a 1:1 mixture of 2.3 M sucrose and 2% methyl cellulose. Grids were viewed using a transmission electron microscope (model 1010; JEOL).

DNA manipulations
Construction of a vector expressing GFP-tagged YLR404wp. A 1.4-kb fragment containing the native promoter and the coding sequence before the stop codon of YLR404W was amplifi ed by PCR using a 5 ' primer (5 ' -HindIII-TTACCATGCACGTTGTCG) and a 3 ' primer (5 ' -BamHI-GCTATGTTTCTTG-GATT). This fragment was then subcloned into the HindIII-and BamHI-cleaved YCplac111-GFP plasmid, in which the GFP coding sequence was inserted between the BamHI and EcoRI restriction sites. A sequence encoding yeast codon bias GFP (yEGFP; a gift from B. Winsor, Universit é Louis Pasteur, Strasbourg, France) was used for all GFP constructs.
Construction of a vector expressing GFP-tagged Tgl3p. A 2.7-kb fragment containing the natural promoter and the coding sequence before the stop codon of TGL3 was amplifi ed by PCR using a 5 ' primer (5 ' -ACG-GC-HindIII-TCTGTT) and a 3 ' primer (5 ' -BamHI-CCTACTCCGTCTTGCTCTT). This fragment was then subcloned into the HindIII-and BamHI-cleaved YCplac111-GFP plasmid, in which the GFP coding sequence was inserted between the BamHI and EcoRI restriction sites.

' -SphI-A T G A T A C A T C A A A G A A G A G A AGCTGG)
and a 3 ' primer (5 ' -XbaI-TCAGGAACTGGAGCAGGTCG G G CGTTG) fatty acid incorporation into all major phospholipids as a result of the deletion of FLD1 ( Fig. 5 and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1).
We identifi ed seipin (Fld1p) as a novel regulator of the cellular dynamics of LDs. The deletion of FLD1 causes increased levels of neutral lipids, clustering of LDs, and formation of enlarged (supersized) LDs. Increased neutral lipids often lead to an increase in the number but not the size of LDs ( Fig. 1, C ,b and c;and Fig. S2, available at http://www.jcb.org/cgi/content/ full/jcb.200711136/DC1). Therefore, the appearance of supersized LDs in fl d1 ⌬ cells is unlikely to be caused by an increase in neutral lipids. Rather, the physical property of the surface of LDs might have been altered in fl d1 ⌬ cells as a result of changes in phospholipids, which may facilitate the clustering and fusion of LDs. In support of this, LDs isolated from fl d1 ⌬ cells can aggregate and fuse without the supply of ATP and cytoplasmic proteins ( Fig. 3 C ).
What is the molecular function of Fld1p or seipin? We favor a role of Fld1p in phospholipid/fatty acid metabolism for the following reasons: (1) a shift from long to medium/short acyl chains was detected in fl d1 ⌬ cells ( Fig. 5 ); (2) LDs isolated from fl d1 ⌬ cells can aggregate and fuse without the supply of ATP and cytoplasmic proteins, suggesting a role for phospholipids ( Fig. 3 ); (3) the other gene that is associated with CGL is APGAT2, a major enzyme in phospholipid metabolism; and (4) lipin, another well-known protein that is associated with lipodystrophy in mice, is a phosphatidate phosphatase . Deletion of SPO7 or NEM1 , two genes involved in the activation of lipin, also caused aberrant LD morphology (Table S1 and our unpublished data).
Results described herein show for the fi rst time that seipin and its homologues modulate the formation and especially fusion of the LDs. Our data also suggest that changes in phospholipid metabolism may be the unifying theme for both CGL1 and CGL2. Understanding the molecular function of seipin will provide mechanistic insights into LD formation and adipogenesis.

Media
Unless otherwise stated, yeast cells were grown with rotary shaking at 30 ° C in liquid YPD medium (1% yeast extract, 2% bacto peptone, and 2% dextrose). Alternatively, cells were grown in SC medium (0.67% nitrogen base and 2% dextrose with all amino acids supplemented), YPO medium (1% yeast extract, 2% bacto peptone, and 0.1% oleic acid in 1% Brij58), or in YPDO medium (1% yeast extract, 2% bacto peptone, 2% dextrose, and 0.1% oleic acid in 1% Brij58). Plasmid-carrying strains were grown on synthetic medium with appropriate selection as described previously ( Kaiser et al., 1994 ). Cell fractionation Preparation of spheroplasts. Cells were harvested by centrifugation at 3,000 g for 5 min, washed once with double-distilled H 2 O, suspended to 0.5 g of wet weight/ml in 0.1 M Tris-Cl, pH 9.4, and 10 mM DTT, and incubated at 30 ° C for 10 min. Afterward, they were washed once with 1.2 M sorbitol and suspended in 1.2 M sorbitol and 20 mM K 3 PO 4 , pH 7.4, to 0.15 g of wet weight/ml. Zymose 20T was added to 2 mg/g of wet weight cells. The suspension was incubated at 30 ° C with gentle shaking for 20 -60 min. Spheroplasts were harvested by centrifugation for 5 min at 1,500 g , washed twice with 1.2 M sorbitol, and suspended in the appropriate buffer for homogenization.
Differential centrifugation and sucrose density gradient analysis of whole cell extract. Spheroplasts were lysed in ice-cold lysis buffer II (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 0.2 M sorbitol) containing protease inhibitors. Cell lysates were cleared at 500 g for 5 min, resulting in supernatant S5, which was then subjected to centrifugation at 13,000 g for 15 min, resulting in supernatant S13 and pellet P13. A continuous sucrose density gradient fractionation procedure was used as described previously ( K ö lling and Hollenberg, 1994 ) except that only 13 fractions were taken from top to bottom. In brief, cells were lysed in STED10 (10% wt/wt sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA, and 1 mM DTT) using a glass bead beater and were spun at 500 g for 5 min to remove cell debris. 950 l of cleared cell extract was loaded onto a sucrose density prepared as follows: in 13-ml centrifuge tubes (SW41; Beckman Coulter), 3.8 ml STED53 (53% sucrose), STED35 (35% sucrose), and STED20 (20% sucrose) was loaded on top of each other. Centrifugation was performed at 30,000 rpm for 13 -17 h for membranes to reach their equilibrium density. 950-l fractions were collected from the top of the gradient. Dpm1p was used as an ER marker.
Isolation of LDs. LDs were purifi ed as described by Leber et al. (1994) with minor modifi cations. In brief, 0.15 g/ml spheroplasts were suspended in breaking buffer (10 mM Tris-Cl, pH 6.9, 0.2 mM EDTA, and 12% [wt/wt] Ficoll 400) and chilled on ice. Protease inhibitors were added in buffers as follows: 1 mM PMSF, 2 μ g/ml aprotinin, 0.5 μ g/ml leupeptine, and 1 μ g/ml pepstatin A. Spheroplasts were homogenized with a Dounce homogenizer applying 10 -20 strokes using a loose-fi tting pestle, and the homogenate was transferred into 13.5-ml centrifuge tubes (Ultra-Clear; Beckman Coulter) and overlaid with an equal volume of breaking buffer. Centrifugation was performed for 60 min at 28,000 rpm in an SW41 swing bucket rotor (Beckman Coulter). A fl oating layer that consists mainly of LDs and vacuoles was collected and suspended gently in breaking buffer using a Dounce homogenizer with a loose-fi tting pestle. The suspension was again transferred into 13.5-ml tubes, overlaid with an equal volume of 10 mM Tris-Cl, pH 7.4, 0.2 mM EDTA, and 8% (wt/wt) Ficoll 400, and centrifuged for 60 min at 28,000 rpm in an SW41 swing bucket rotor. The fl oating layer was recovered, suspended gently in 10 mM Tris-Cl, 0.2 mM EDTA, 0.6 M sorbitol, and 8% (wt/wt) Ficoll 400 in 13.5-ml tubes, and overlaid with 10 mM Tris-Cl, 0.2 mM EDTA, and 0.25 M sorbitol. After centrifugation at 28,000 rpm for 30 min, the fl oating layer consists of highly enriched LDs that are separated from vacuoles.

Online supplemental material
Tables S1 and S2 summarize the genes identifi ed in the genome-wide screen for fl d strains and mld strains, respectively. Fig. S1 presents neutral lipid analysis of wild-type and ylr404w ⌬ ( fl d1 ⌬ ) strains. Fig. S2 shows that mutants ( mld ) with increased levels of neutral lipids do not contain enlarged LDs. Fig. S3 is an analysis of phosphatidylethanolamine and phosphatidylcholine, which is continued from Fig. 5 . Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200711136/DC1.