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

Fei W, Shui G, Gaeta B, Du X, Kuerschner L, Li P, Brown AJ, Wenk MR, Parton RG, Yang H - J. Cell Biol. (2008)

Bottom Line: Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Delta 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 fld1Delta cells.These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore.

ABSTRACT
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 approximately 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 fld1Delta 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 fld1Delta cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Delta cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

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Fld1p localizes predominantly to the ER. (A) The expression of FLD1-GFP complements the fld1Δ phenotype. Cells were transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP and grown in SC media without leucine. (B) fld1Δ cells transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP were lysed and immunoblotted with anti-GFP or Dpm1p antisera. (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 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, fixed, and processed for immuno-EM with antisera against GFP. (a) An overview of a representative cell. (b) High magnification image depicting immunoreactivity at the cortical ER. (c and d) High magnification images depicting immunoreactivity at the cortical ER (arrowheads) and in close proximity to LDs (arrows). Vac, vacuole; ld, lipid droplet. Bars: (A and E) 5 μm; (F, a) 1 μm; (F, b–d) 200 nm.
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fig2: Fld1p localizes predominantly to the ER. (A) The expression of FLD1-GFP complements the fld1Δ phenotype. Cells were transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP and grown in SC media without leucine. (B) fld1Δ cells transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP were lysed and immunoblotted with anti-GFP or Dpm1p antisera. (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 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, fixed, and processed for immuno-EM with antisera against GFP. (a) An overview of a representative cell. (b) High magnification image depicting immunoreactivity at the cortical ER. (c and d) High magnification images depicting immunoreactivity at the cortical ER (arrowheads) and in close proximity to LDs (arrows). Vac, vacuole; ld, lipid droplet. Bars: (A and E) 5 μm; (F, a) 1 μm; (F, b–d) 200 nm.

Mentions: The expression of FLD1-GFP in fld1Δ 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 fld1Δ 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 fluorescent 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).


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

Fei W, Shui G, Gaeta B, Du X, Kuerschner L, Li P, Brown AJ, Wenk MR, Parton RG, Yang H - J. Cell Biol. (2008)

Fld1p localizes predominantly to the ER. (A) The expression of FLD1-GFP complements the fld1Δ phenotype. Cells were transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP and grown in SC media without leucine. (B) fld1Δ cells transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP were lysed and immunoblotted with anti-GFP or Dpm1p antisera. (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 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, fixed, and processed for immuno-EM with antisera against GFP. (a) An overview of a representative cell. (b) High magnification image depicting immunoreactivity at the cortical ER. (c and d) High magnification images depicting immunoreactivity at the cortical ER (arrowheads) and in close proximity to LDs (arrows). Vac, vacuole; ld, lipid droplet. Bars: (A and E) 5 μm; (F, a) 1 μm; (F, b–d) 200 nm.
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fig2: Fld1p localizes predominantly to the ER. (A) The expression of FLD1-GFP complements the fld1Δ phenotype. Cells were transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP and grown in SC media without leucine. (B) fld1Δ cells transformed either with YCplac111 vector alone or with YCplac111-FLD1-GFP were lysed and immunoblotted with anti-GFP or Dpm1p antisera. (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 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, fixed, and processed for immuno-EM with antisera against GFP. (a) An overview of a representative cell. (b) High magnification image depicting immunoreactivity at the cortical ER. (c and d) High magnification images depicting immunoreactivity at the cortical ER (arrowheads) and in close proximity to LDs (arrows). Vac, vacuole; ld, lipid droplet. Bars: (A and E) 5 μm; (F, a) 1 μm; (F, b–d) 200 nm.
Mentions: The expression of FLD1-GFP in fld1Δ 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 fld1Δ 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 fluorescent 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).

Bottom Line: Cells lacking FLD1 contain strikingly enlarged (supersized) LDs, and LDs from fld1Delta 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 fld1Delta cells.These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore.

ABSTRACT
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 approximately 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 fld1Delta 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 fld1Delta cells. Lipid profiling reveals alterations in acyl chain compositions of major phospholipids in fld1Delta cells. These results suggest that an evolutionally conserved function of seipin in phospholipid metabolism and LD formation may be functionally important in human adipogenesis.

Show MeSH
Related in: MedlinePlus