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.

Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris.

Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris, which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae. Using P. pastoris psd1Delta and psd2Delta mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP)-ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris, which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes.

Phosphorylation of different tau sites during progression of Alzheimer's disease.

Alzheimer's disease is characterized by accumulation of amyloid plaques and tau aggregates in several cortical brain regions. Tau phosphorylation causes formation of neurofibrillary tangles and neuropil threads. Phosphorylation at tau Ser202/Thr205 is well characterized since labeling of this site is used to assign Braak stage based on occurrence of neurofibrillary tangles. Only little is known about the spatial and temporal phosphorylation profile of other phosphorylated tau (ptau) sites. Here, we investigate total tau and ptau at residues Tyr18, Ser199, Ser202/Thr205, Thr231, Ser262, Ser396, Ser422 as well as amyloid-ß plaques in human brain tissue of AD patients and controls. Allo- and isocortical brain regions were evaluated applying rater-independent automated quantification based on digital image analysis. We found that the level of ptau at several residues, like Ser199, Ser202/Thr205, and Ser422 was similar in healthy controls and Braak stages I to IV but was increased in Braak stage V/VI throughout the entire isocortex and transentorhinal cortex. Quantification of ThioS-stained plaques showed a similar pattern. Only tau phosphorylation at Tyr18 and Thr231 was already significantly increased in the transentorhinal region at Braak stage III/IV and hence showed a progressive increase with increasing Braak stages. Additionally, the increase in phosphorylation relative to controls was highest at Tyr18, Thr231 and Ser199. By contrast, Ser396 tau and Ser262 tau showed only a weak phosphorylation in all analyzed brain regions and only minor progression. Our results suggest that the ptau burden in the isocortex is comparable between all analyzed ptau sites when using a quantitative approach while levels of ptau at Tyr18 or Thr231 in the transentorhinal region are different between all Braak stages. Hence these sites could be crucial in the pathogenesis of AD already at early stages and therefore represent putative novel therapeutic targets.

At4g24160, a soluble acyl-coenzyme A-dependent lysophosphatidic acid acyltransferase.

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the alpha/beta-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX(4)D acyltransferase motif, and V(X)(3)HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.