Neutral lipid storage and mobilization

In the yeast as in most other cells neutral lipids TAG (triacylglycerols) and SE (steryl esters) are stored in subcellular structures named lipid particles/droplets (LP). Upon requirement, TAG and SE can be mobilized and serve as building blocks for membrane biosynthesis. In this project, we investigate and characterize enzymatic steps which lead to the storage of TAG and SE and at the same time to the biogenesis of lipid particles. Moreover, we study processes involved in the mobilization of TAG and SE depots.
 
Prerequisite for the understanding of LP function and structure is elucidation of their molecular equipment. For this purpose, we performed conventional analysis and mass spectrometric analysis of lipids (TAG, STE, phospholipids), but also of proteins which are present on the surface of LP. These analyses were carried out with LP from cells grown on glucose or oleic acid. Results obtained by these methods revealed marked differences in the lipidome, but also in the proteome of LP isolated from yeast grown under different conditions. Moreover, proteome analysis of LP led to identification of several new putative LP proteins.
 
In the yeast, the SE synthases Are1p and Are2p, and the TAG synthases Dga1p and Lro1p contribute to the formation of lipid depots. Triple mutants with only one of these four enzymes active were used to obtain detailed information about the coordinate process of neutral lipid synthesis and the specific contribution of each of the four acyltransferases to LP biogenesis. All four triple mutants with only one of the gene products, Dga1p, Lro1p, Are1p or Are2p, active form LP, although at different rate and lipid composition. Through these experiments we showed that TAG or SE alone are sufficient for LP biogenesis. Biophysical methods revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the droplets (Figure 2).


Figure 2: Internal structure of yeast lipid particles (LP).

 

A core of TAG is surrounded by several shells of SE. The surface of LP consists of a phospholipid monolayer with proteins embedded.

 

Similar to TAG and SE synthesizing enzymes, proteins involved in degradation of neutral lipids occur in redundancy. Three TAG lipases named Tgl3p, Tgl4p and Tgl5p, and three SE hydrolases named Yeh1p, Yeh2p and Tgl1p were identified in the yeast. Recently, we studied enzymatic properties of the three SE hydrolytic enzymes in some detail. Our investigations demonstrated that each SE hydrolase exhibits certain substrate specificity. We also demonstrated that sterol intermediates stored as SE are readily mobilized through SE hydrolases, recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol.

 

Previous work from our laboratory had demonstrated that deletion of TGL3, encoding the major yeast TAG lipase located to LP, resulted in a decreased mobilization of TAG. TAG stored in LP of a tgl3Δ mutant contained slightly increased amounts of 22:0 and 26:0 very long chain fatty acids (VLCFAs). These VLCFAs are indispensable for sphingolipid biosynthesis and crucial for raft association in the yeast. Recent experiments showed that tgl mutants have a significantly reduced rate of sphingolipid biosynthesis, but also of phospholipid synthesis. We hypothesize that stored TAG can be a fatty acid donor for sphingolipid and phospholipid formation and TAG lipases contribute to this pathway.

 

Another potential non-polar storage lipid is squalene. In the yeast, the amount of squalene can be increased by varying culture conditions or by genetic manipulation. As an example, in strains deleted of HEM1 squalene accumulates and is stored mainly in LP. Interestingly, a heme deficient dga1Δlro1Δare1Δare2Δ quadruple mutant (Qhem1Δ) which is devoid of the classical storage lipids, TAG and SE, accumulates substantial amounts of squalene in cellular membranes, especially in microsomes and the plasma membrane. The fact that Qhem1Δ does not form LP suggests that biogenesis of these storage particles cannot be initiated by squalene which is synthesized in the ER similar to TAG and SE. This result also indicates that squalene accumulation, at least under the conditions tested, is not lipotoxic. Finally, our experiments demonstrate that squalene incorporated into organelle membranes does not compromise cellular function.