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Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.

Cartwright BR, Binns DD, Hilton CL, Han S, Gao Q, Goodman JM - Mol. Biol. Cell (2014)

Bottom Line: Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology.Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant.We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.

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In the absence of seipin, TG accumulates in the ER during de novo LD formation. 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells were induced in rich galactose medium for 6 h, lysed, and fractionated. (A and C) Enriched ER membranes were centrifuged through a sucrose gradient, and fractions were blotted with α-Dpm1p (ER) or α-porin antibody. “Both” indicates a mixture of isolated membranes (4:1 3KO(GALDGA1) to 3KO(GALDGA1)fld1∆ membranes by protein) from both strains; “wt” indicates the parental W303-1A strain. Representative blots for seipin from one of three independent experiments shown for 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells, and one of two independent experiments for porin. (B) Droplets and membranes from cells expressing Erg6-mRed and Sec63-GFP were separated and probed with α-Dpm1p or α-GFP; identical percents of LD and membrane fraction were analyzed. Parallel samples from cells expressing Erg6-DsRed were probed with α-DsRed. (Sec63-GFP interfered with the α-Erg6p signal.) Dpm1p is exclusively in the membrane fraction. (D) TG levels in the PNS fractions are compared in the strains indicated. (E) TG levels in the 3KO(GALDGA1) strain are compared with the 3KO(GALDGA1)fld1∆ strain in isolated droplets and enriched ER. The results of two experiments are shown in which organelles from the two strains were prepared in parallel. “100%” signifies the ratio of TG in the LD or ER fraction relative to TG in the PNS in the 3KO(GALDGA1) strain. Absolute values of TG (expressed as micrograms of trioleoyl glycerol equivalents) in the PNS and purified LDs are 697 and 88.9, respectively, in one experiment, and 263 and 52.4, respectively, for the other. Absolute values in the PNS and purified ER are 377 and 19.2, respectively, for one experiment, and 288 and 25.2, respectively, for the other. Comparison of these values with those from the 3KO(GALDGA1)fld1∆ strains reveals 36.5 and 40.2% (in the two experiments) as much TG in LDs from the seipin-minus strain, and 144 and 143% as much TAG in ER from the seipin-minus strain.
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Figure 2: In the absence of seipin, TG accumulates in the ER during de novo LD formation. 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells were induced in rich galactose medium for 6 h, lysed, and fractionated. (A and C) Enriched ER membranes were centrifuged through a sucrose gradient, and fractions were blotted with α-Dpm1p (ER) or α-porin antibody. “Both” indicates a mixture of isolated membranes (4:1 3KO(GALDGA1) to 3KO(GALDGA1)fld1∆ membranes by protein) from both strains; “wt” indicates the parental W303-1A strain. Representative blots for seipin from one of three independent experiments shown for 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells, and one of two independent experiments for porin. (B) Droplets and membranes from cells expressing Erg6-mRed and Sec63-GFP were separated and probed with α-Dpm1p or α-GFP; identical percents of LD and membrane fraction were analyzed. Parallel samples from cells expressing Erg6-DsRed were probed with α-DsRed. (Sec63-GFP interfered with the α-Erg6p signal.) Dpm1p is exclusively in the membrane fraction. (D) TG levels in the PNS fractions are compared in the strains indicated. (E) TG levels in the 3KO(GALDGA1) strain are compared with the 3KO(GALDGA1)fld1∆ strain in isolated droplets and enriched ER. The results of two experiments are shown in which organelles from the two strains were prepared in parallel. “100%” signifies the ratio of TG in the LD or ER fraction relative to TG in the PNS in the 3KO(GALDGA1) strain. Absolute values of TG (expressed as micrograms of trioleoyl glycerol equivalents) in the PNS and purified LDs are 697 and 88.9, respectively, in one experiment, and 263 and 52.4, respectively, for the other. Absolute values in the PNS and purified ER are 377 and 19.2, respectively, for one experiment, and 288 and 25.2, respectively, for the other. Comparison of these values with those from the 3KO(GALDGA1)fld1∆ strains reveals 36.5 and 40.2% (in the two experiments) as much TG in LDs from the seipin-minus strain, and 144 and 143% as much TAG in ER from the seipin-minus strain.

Mentions: To test the accumulation of TG in membranes biochemically, we harvested cells grown in galactose for 6 h. A membrane fraction was prepared and subjected to isopycnic centrifugation through a 1.0–1.9 M sucrose gradient, and fractions were then probed by immunoblotting for Dpm1p, an integral ER membrane marker (Orlean et al., 1988). ER from the seipin-positive strain migrated to 1.47 M sucrose, while the bulk of membranes from the seipin-negative strain migrated lighter, to 1.33 M sucrose, consistent with the accumulation of NL in the ER in the absence of seipin (Figure 2A). Although a singular report indicated a partial localization of Dpm1p to droplets under some conditions (Takeda and Nakano, 2008), we failed to observe more than a trace (∼1%) of Dpm1p in droplets in our system (Figure 2B; Erg6-DsRed and Sec63–green fluorescent protein [Sec63-GFP] were used as markers of droplets and ER, respectively). Mitochondria were also shifted to a lower density but to a smaller extent (Figure 2C), suggesting that the accumulated NL was not limited to the ER.


Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.

Cartwright BR, Binns DD, Hilton CL, Han S, Gao Q, Goodman JM - Mol. Biol. Cell (2014)

In the absence of seipin, TG accumulates in the ER during de novo LD formation. 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells were induced in rich galactose medium for 6 h, lysed, and fractionated. (A and C) Enriched ER membranes were centrifuged through a sucrose gradient, and fractions were blotted with α-Dpm1p (ER) or α-porin antibody. “Both” indicates a mixture of isolated membranes (4:1 3KO(GALDGA1) to 3KO(GALDGA1)fld1∆ membranes by protein) from both strains; “wt” indicates the parental W303-1A strain. Representative blots for seipin from one of three independent experiments shown for 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells, and one of two independent experiments for porin. (B) Droplets and membranes from cells expressing Erg6-mRed and Sec63-GFP were separated and probed with α-Dpm1p or α-GFP; identical percents of LD and membrane fraction were analyzed. Parallel samples from cells expressing Erg6-DsRed were probed with α-DsRed. (Sec63-GFP interfered with the α-Erg6p signal.) Dpm1p is exclusively in the membrane fraction. (D) TG levels in the PNS fractions are compared in the strains indicated. (E) TG levels in the 3KO(GALDGA1) strain are compared with the 3KO(GALDGA1)fld1∆ strain in isolated droplets and enriched ER. The results of two experiments are shown in which organelles from the two strains were prepared in parallel. “100%” signifies the ratio of TG in the LD or ER fraction relative to TG in the PNS in the 3KO(GALDGA1) strain. Absolute values of TG (expressed as micrograms of trioleoyl glycerol equivalents) in the PNS and purified LDs are 697 and 88.9, respectively, in one experiment, and 263 and 52.4, respectively, for the other. Absolute values in the PNS and purified ER are 377 and 19.2, respectively, for one experiment, and 288 and 25.2, respectively, for the other. Comparison of these values with those from the 3KO(GALDGA1)fld1∆ strains reveals 36.5 and 40.2% (in the two experiments) as much TG in LDs from the seipin-minus strain, and 144 and 143% as much TAG in ER from the seipin-minus strain.
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Figure 2: In the absence of seipin, TG accumulates in the ER during de novo LD formation. 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells were induced in rich galactose medium for 6 h, lysed, and fractionated. (A and C) Enriched ER membranes were centrifuged through a sucrose gradient, and fractions were blotted with α-Dpm1p (ER) or α-porin antibody. “Both” indicates a mixture of isolated membranes (4:1 3KO(GALDGA1) to 3KO(GALDGA1)fld1∆ membranes by protein) from both strains; “wt” indicates the parental W303-1A strain. Representative blots for seipin from one of three independent experiments shown for 3KO(GALDGA1) and 3KO(GALDGA1)fld1∆ cells, and one of two independent experiments for porin. (B) Droplets and membranes from cells expressing Erg6-mRed and Sec63-GFP were separated and probed with α-Dpm1p or α-GFP; identical percents of LD and membrane fraction were analyzed. Parallel samples from cells expressing Erg6-DsRed were probed with α-DsRed. (Sec63-GFP interfered with the α-Erg6p signal.) Dpm1p is exclusively in the membrane fraction. (D) TG levels in the PNS fractions are compared in the strains indicated. (E) TG levels in the 3KO(GALDGA1) strain are compared with the 3KO(GALDGA1)fld1∆ strain in isolated droplets and enriched ER. The results of two experiments are shown in which organelles from the two strains were prepared in parallel. “100%” signifies the ratio of TG in the LD or ER fraction relative to TG in the PNS in the 3KO(GALDGA1) strain. Absolute values of TG (expressed as micrograms of trioleoyl glycerol equivalents) in the PNS and purified LDs are 697 and 88.9, respectively, in one experiment, and 263 and 52.4, respectively, for the other. Absolute values in the PNS and purified ER are 377 and 19.2, respectively, for one experiment, and 288 and 25.2, respectively, for the other. Comparison of these values with those from the 3KO(GALDGA1)fld1∆ strains reveals 36.5 and 40.2% (in the two experiments) as much TG in LDs from the seipin-minus strain, and 144 and 143% as much TAG in ER from the seipin-minus strain.
Mentions: To test the accumulation of TG in membranes biochemically, we harvested cells grown in galactose for 6 h. A membrane fraction was prepared and subjected to isopycnic centrifugation through a 1.0–1.9 M sucrose gradient, and fractions were then probed by immunoblotting for Dpm1p, an integral ER membrane marker (Orlean et al., 1988). ER from the seipin-positive strain migrated to 1.47 M sucrose, while the bulk of membranes from the seipin-negative strain migrated lighter, to 1.33 M sucrose, consistent with the accumulation of NL in the ER in the absence of seipin (Figure 2A). Although a singular report indicated a partial localization of Dpm1p to droplets under some conditions (Takeda and Nakano, 2008), we failed to observe more than a trace (∼1%) of Dpm1p in droplets in our system (Figure 2B; Erg6-DsRed and Sec63–green fluorescent protein [Sec63-GFP] were used as markers of droplets and ER, respectively). Mitochondria were also shifted to a lower density but to a smaller extent (Figure 2C), suggesting that the accumulated NL was not limited to the ER.

Bottom Line: Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology.Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant.We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.

Show MeSH
Related in: MedlinePlus