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 dissect the role of seipin in LD formation, we screened a small panel of seipin-deletion mutants for defects in steady-state LD morphology (unpublished data). Most remarkable of these was a deletion of 14 amino acids (MKINVSRPLQFLQW), comprising the amino terminal domain that faces the cytosol and precedes the first membrane span. While the seipin- strain is characterized by droplets of widely varying size, especially when cultured in medium containing oleic acid (Szymanski et al., 2007), cells expressing the N-terminal deletion mutant (fld1∆Nterm) from a plasmid in the background resulted in a remarkable prominence of SLDs (Figure 3A). This is consistent with a recently reported phenotype of fewer, larger droplets for a 10–amino acid N-terminal deletion of seipin, which was not characterized further (Wang et al., 2014). The fld1∆Nterm mutant produced fewer droplets per cell (Figure 3B) and more cells with SLDs (defined as LDs with >1-μm diameter by Yang; Fei et al., 2011a; Figure 3C) than both wild-type and seipin knockout cells. The overexpressed N-terminal deletion mutant protein both localized to the ER and formed high-molecular-weight complexes (Supplemental Figure S8), similar to wild-type. An SLD phenotype was also observed for endogenously expressed fld1∆Nterm integrated at the genomic seipin locus (Supplemental Figure S9A). Fluorescently tagged fld1∆Nterm forms several puncta similar to wild-type. Whereas wild-type seipin clearly localizes at or adjacent to the several droplets in cells, most puncta from fld1∆Nterm were dispersed in the cell, with at least one seipin spot associated with each of the few supersized droplets. (Supplemental Figure S9B).
Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041.