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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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Expression of human and mouse BSCL2 (seipin) rescues defects in LD morphology in fld1Δ cells. (A) Sequence alignment of seipin homologues with gene identification numbers. 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 secondary structures predicted by PSIPRED (position-specific 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 (McGuffin et al., 2000). (B and C) The expression of seipin in yeast. fld1Δ strains transformed with YCplac111-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 fluorescence microscopy. Bars, 5 μm.
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fig4: Expression of human and mouse BSCL2 (seipin) rescues defects in LD morphology in fld1Δ cells. (A) Sequence alignment of seipin homologues with gene identification numbers. 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 secondary structures predicted by PSIPRED (position-specific 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 (McGuffin et al., 2000). (B and C) The expression of seipin in yeast. fld1Δ strains transformed with YCplac111-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 fluorescence microscopy. Bars, 5 μm.

Mentions: 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) and motorneuron disorders, was identified (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 fld1Δ cells grown in SC media. The average diameter of the LDs is 1.27 ± 0.19 μm (n = 117) without human seipin and 0.43 ± 0.05 μm (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). This is not surprising because A212P is considered a loss-of-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.


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)

Expression of human and mouse BSCL2 (seipin) rescues defects in LD morphology in fld1Δ cells. (A) Sequence alignment of seipin homologues with gene identification numbers. 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 secondary structures predicted by PSIPRED (position-specific 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 (McGuffin et al., 2000). (B and C) The expression of seipin in yeast. fld1Δ strains transformed with YCplac111-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 fluorescence microscopy. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2234226&req=5

fig4: Expression of human and mouse BSCL2 (seipin) rescues defects in LD morphology in fld1Δ cells. (A) Sequence alignment of seipin homologues with gene identification numbers. 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 secondary structures predicted by PSIPRED (position-specific 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 (McGuffin et al., 2000). (B and C) The expression of seipin in yeast. fld1Δ strains transformed with YCplac111-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 fluorescence microscopy. Bars, 5 μm.
Mentions: 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) and motorneuron disorders, was identified (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 fld1Δ cells grown in SC media. The average diameter of the LDs is 1.27 ± 0.19 μm (n = 117) without human seipin and 0.43 ± 0.05 μm (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). This is not surprising because A212P is considered a loss-of-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.

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