Karlheinz Grillitsch

Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: lipidome meets proteome.

In the yeast Saccharomyces cerevisiae as in other eukaryotes non-polar lipids are a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerols (TG) and steryl esters (SE) are stored in so-called lipid particles/droplets (LP) as biologically inert form of fatty acids and sterols. To understand LP structure and function in more detail we investigated the molecular equipment of this compartment making use of mass spectrometric analysis of lipids (TG, SE, phospholipids) and proteins. We addressed the question whether or not lipid and protein composition of LP influence each other and performed analyses of LP from cells grown on two different carbon sources, glucose and oleate. Growth of cells on oleate caused dramatic cellular changes including accumulation of TG at the expense of SE, enhanced the amount of glycerophospholipids and strongly increased the degree of unsaturation in all lipid classes. Most interestingly, oleate as a carbon source led to adaptation of the LP proteome resulting in the appearance of several novel LP proteins. Localization of these new LP proteins was confirmed by cell fractionation. Proteomes of LP variants from cells grown on glucose or oleate, respectively, were compared and are discussed with emphasis on the different groups of proteins detected through this analysis. In summary, we demonstrate flexibility of the yeast LP lipidome and proteome and the ability of LP to adapt to environmental changes.

Synthesis and turnover of non-polar lipids in yeast.

In the yeast Saccharomyces cerevisiae as in other eukaryotic cells non-polar lipids form a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerol (TAG) and steryl ester (STE), are synthesized by enzymes with overlapping function. Recently, genes encoding these enzymes were identified and gene products were partially characterized. Once formed, TAG and STE are stored in so-called lipid particles/droplets. This compartment which is reminiscent of mammalian lipoproteins from the structural viewpoint is, however, not only a lipid depot but also an organelle actively contributing to lipid metabolism. Non-polar lipid degrading enzymes, TAG lipases and STE hydrolases, also occur in redundancy in the yeast. These proteins, which are components of the lipid particle surface membrane with the exception of one plasma membrane localized STE hydrolase, mobilize non-polar lipids upon requirement. In this review, we describe the coordinate pathways of non-polar lipid synthesis, storage and mobilization in yeast with special emphasis on the role of the different enzymes and organelles involved in these processes. Moreover, we will discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae

In the yeast as in other eukaryotes, formation and hydrolysis of steryl esters (SE) are processes linked to lipid storage. In Saccharomyces cerevisiae, the three SE hydrolases Tgl1p, Yeh1p and Yeh2p contribute to SE mobilization from their site of storage, the lipid particles/droplets. Here, we provide evidence for enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrate that each SE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in SE mobilization also affects sterol biosynthesis in a type of feedback regulation. Sterol intermediates stored in SE and set free by SE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the endoplasmic reticulum, where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors are randomly distributed within the cell after cleavage from SE. Conclusively, SE storage and mobilization although being dispensable for yeast viability contribute markedly to sterol homeostasis and distribution.

Analysis of Lipid Particles from Yeast

Quantitative analysis of components from different subcellular fractions is a key to the understanding of metabolic function as well as to the origin, the biogenesis, and the crosstalk of organelles. The yeast is an excellent model organism to address such questions from the biochemical, molecular biological, and cell biological viewpoints. A yeast organelle which gained much interest during the last decade is the lipid particle/droplet (LP), a storage compartment for nonpolar lipids but at the same time an organelle actively contributing to cellular metabolism. In this chapter, we describe methods and techniques that are commonly used to analyze lipids from LP at the molecular level by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry. We provide an easy to follow guideline for the isolation of these organelles, the qualitative and quantitative analysis of lipid components and show results obtained with these methods.

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.

Modulation of sterol homeostasis by the Cdc42p effectors Cla4p and Ste20p in the yeast Saccharomyces cerevisiae: Sterol homeostasis modulation by Ste20p and Cla4p

The conserved Rho‐type GTPase Cdc42p is a key regulator of signal transduction and polarity in eukaryotic cells. In the yeast Saccharomyces cerevisiae, Cdc42p promotes polarized growth through the p21‐activated kinases Ste20p and Cla4p. Previously, we demonstrated that Ste20p forms a complex with Erg4p, Cbr1p and Ncp1p, which all catalyze important steps in sterol biosynthesis. CLA4 interacts genetically with ERG4 and NCP1. Furthermore, Erg4p, Ncp1p and Cbr1p play important roles in cell polarization during vegetative growth, mating and filamentation. As Ste20p and Cla4p are involved in these processes it seems likely that sterol biosynthetic enzymes and p21‐activated kinases act in related pathways. Here, we demonstrate that the deletion of either STE20 or CLA4 results in increased levels of sterols. In addition, higher concentrations of steryl esters, the storage form of sterols, were observed in cla4Δ cells. CLA4 expression from a multicopy plasmid reduces enzyme activity of Are2p, the major steryl ester synthase, under aerobic conditions. Altogether, our data suggest that Ste20p and Cla4p may function as negative modulators of sterol biosynthesis. Moreover, Cla4p has a negative effect on steryl ester formation. As sterol homeostasis is crucial for cell polarization, Ste20p and Cla4p may regulate cell polarity in part through the modulation of sterol homeostasis.

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris.

In yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1∆lro1∆ double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae.

Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.

The effect of hypoxia on the lipidome of recombinant Pichia pastoris

BackgroundCultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia.ResultsTo improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition.ConclusionsOur results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.