Seipin is involved in the regulation of phosphatidic acid metabolism at a subdomain of the nuclear envelope in yeast

Abstract

Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1-Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1-Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site.

Lipid synthesis and membrane contact sites: a crossroads for cellular physiology

“…Deletion of the CHO2 gene, which decreases PC synthesis through the methylation pathway and whose inactivation also results in abnormal LD morphology, prevented the inositol-induced formation of ER-entangled LD aggregates (25,51,59), supporting the idea that augmented PL synthesis contributes to the change of LD morphology observed in seipin complex mutants upon inositol supplementation. This idea was further supported whereby overexpression of the CDP-DAG synthase encoded by CDS1, activation upon cytosol acidification have not been solved: mere acidification could activate the phosphatase or it could be mediated by TORC, as it is known that cytosol acidification upon glucose exhaustion activates TORC, and that Nem1/Spo7 is a downstream target of this kinase (49).…”

“…LDs from seipin complex mutant cells are irregular in shape and their PL monolayer is wrinkled around the NL core (21). In addition, the ER-LD contact sites are expanded and bloated (21,25). The assembly of purified Fld1 into a nonamer of toroid form (61), plus the evidence that Ldb16 interacts with Fld1 forming a macromolecular complex of still undefined structure (59), supports the idea of the seipin complex acting as a diffusion barrier at the ER-LD contacts sites.…”

“…Contemporarily with this report, two other groups showed the formation of Opi1 foci associated with LD in seipin complex-deficient cells (24,25). In its membrane bound form, Opi1 interacts with both PA and the ER integral protein, Scs2.…”

“…The absence of Fld1 or Lbd16 elicits similar LD phenotypes, as well as similar perturbations on lipid metabolism. Typically, fld1 and ldb16 cells display a heterogeneous LD phenotype that depends on growth condition (18,20,25,51,59,60). When grown in minimal media, most of the fld1 and ldb16 mutant cells present one or a few SLDs.…”

“…Interestingly, LDs are tightly associated with the ER at MCSs (18)(19)(20)(21), and the activation and membrane recruitment of Pah1 takes place in the vicinity of LDs, suggesting a channeling mechanism of DAG delivery for TAG synthesis and sorting into LDs (22)(23)(24)(25).…”