Seipin performs dissectible functions in promoting lipid droplet biogenesis and regulating droplet morphology.
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.
Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.Show MeSH
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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.
Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.