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

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.

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression.

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle-dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle-dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A(Cdc55)) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.

The emergence of lipid droplets in yeast: current status and experimental approaches.

The 'discovery' of lipid droplets as a metabolically highly active subcellular organelle has sparked great scientific interest in its research in recent years. The previous view of a rather inert storage pool of neutral lipids--triacylglycerol and sterols or steryl esters--has markedly changed. Driven by the endemic dimensions of lipid-associated disorders on the one hand, and the promising biotechnological application to generate oils ('biodiesel') from single-celled organisms on the other, multiple model organisms are exploited in basic and applied research to develop a better understanding of biogenesis and metabolism of this organelle. This article summarizes the current status of LD research in yeast and experimental approaches to obtain insight into the regulatory and structural components driving lipid droplet formation and their physiological and pathophysiological roles in lipid homeostasis.

An iron 13S-lipoxygenase with an α-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.

Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. Fusarium oxysporum) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in F. oxysporum, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in E. coli, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C18-polyunsaturated fatty acids to 13S-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives.