A subcellular proteome atlas of the yeast Komagataella phaffii.

The compartmentalization of metabolic and regulatory pathways is a common pattern of living organisms. Eukaryotic cells are subdivided into several organelles enclosed by lipid membranes. Organelle proteomes define their functions. Yeasts, as simple eukaryotic single cell organisms, are valuable models for higher eukaryotes and frequently used for biotechnological applications. While the subcellular distribution of proteins is well studied in Saccharomyces cerevisiae, this is not the case for other yeasts like Komagataella phaffii (syn. Pichia pastoris). Different to most well-studied yeasts, K. phaffii can grow on methanol, which provides specific features for production of heterologous proteins and as a model for peroxisome biology. We isolated microsomes, very early Golgi, early Golgi, plasma membrane, vacuole, cytosol, peroxisomes and mitochondria of K. phaffii from glucose- and methanol-grown cultures, quantified their proteomes by liquid chromatography-electrospray ionization-mass spectrometry of either unlabeled or tandem mass tag-labeled samples. Classification of the proteins by their relative enrichment, allowed the separation of enriched proteins from potential contaminants in all cellular compartments except the peroxisomes. We discuss differences to S. cerevisiae, outline organelle specific findings and the major metabolic pathways and provide an interactive map of the subcellular localization of proteins in K. phaffii.

TORC1 ensures membrane trafficking of Tat2 tryptophan permease via a novel transcriptional activator Vhr2 in budding yeast.

The target of rapamycin complex 1 (TORC1) protein kinase is activated by nutrients and controls nutrient uptake via the membrane trafficking of various nutrient permeases. However, its molecular mechanisms remain elusive. Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, "lipid rafts", which are critical for intracellular protein sorting. Here we show that a novel target of rapamycin (TOR)-interacting transcriptional activator Vhr2 is required for full expression of some ERG genes for ergosterol biogenesis and for proper sorting of the tryptophan permease Tat2 in budding yeast. Loss of Vhr2 caused sterol biogenesis disturbance and Tat2 missorting. TORC1 activity maintained VHR2 transcript and protein levels, and total sterol levels. Vhr2 was not involved in regulation of the TORC1-downstream protein kinase Npr1, which regulates Tat2 sorting. This study suggests that TORC1 regulates nutrient uptake via sterol biogenesis.

The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol.

The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.

The role of the membrane lipid composition in the oxidative stress tolerance of different wine yeasts.

Oxidative stress is a common stress in yeasts during the stages of the winemaking process in which aerobic growth occurs, and it can modify the cellular lipid composition. The aim of this study was to evaluate the oxidative stress tolerance of two non-conventional yeasts (Torulaspora delbrueckii and Metschnikowia pulcherrima) compared to Saccharomyces cerevisiae. Therefore, their resistance against H2O2, the ROS production and the cellular lipid composition were assessed. The results showed that the non-Saccharomyces yeasts used in this study exhibited higher resistance to H2O2 stress and lower ROS accumulation than Saccharomyces. Regarding the cellular lipid composition, the two non-Saccharomyces species studied here displayed a high percentage of polyunsaturated fatty acids, which resulted in more fluid membranes. This result could indicate that these yeasts have been evolutionarily adapted to have better resistance against the oxidative stress. Furthermore, under external oxidative stress, non-Saccharomyces yeasts were better able to adapt their lipid composition as a defense mechanism by decreasing their percentage of polyunsaturated fatty acids and squalene and increasing their monounsaturated fatty acids.

Melatonin Minimizes the Impact of Oxidative Stress Induced by Hydrogen Peroxide in Saccharomyces and Non-conventional Yeast.

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized from tryptophan by Saccharomyces cerevisiae and non-conventional yeast species. Antioxidant properties have been suggested as a possible role of melatonin in a S. cerevisiae wine strain. However, the possible antioxidant melatonin effect on non-Saccharomyces species and other strains of S. cerevisiae must be evaluated. The aim of this study was to determine the antioxidant capacity of melatonin in eight S. cerevisiae strains and four non-conventional yeasts (Torulaspora delbrueckii, Metschnikowia pulcherrima, Starmerella bacillaris, and Hanseniaspora uvarum). Therefore, the ROS formation, lipid peroxidation, catalase activity, fatty acid composition, and peroxisome proliferation were investigated. The results showed that the presence of melatonin increases peroxisome accumulation and slightly increases the catalase activity. When cells grown in the presence of melatonin were exposed to oxidative stress induced by H2O2, lower ROS accumulation and lipid peroxidation were observed in all tested strains. Therefore, the increased catalase activity that was a consequence of oxidative stress was lower in the presence of melatonin. Moreover, the presence of MEL modulates cell FA composition, increasing oleic and palmitoleic acids and leading to higher UFA/SFA ratios, which have been previously related to a higher tolerance to H2O2. These findings demonstrate that melatonin can act as an antioxidant compound in both S. cerevisiae and non-Saccharomyces yeasts.

Identification and characterization of the mitochondrial membrane sorting signals in phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744-55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.

The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle.

Steryl ester synthesis, storage and hydrolysis: A contribution to sterol homeostasis.

Sterols are essential lipids of all eukaryotic cells, appearing either as free sterols or steryl esters. Besides other regulatory mechanisms, esterification of sterols and hydrolysis of steryl esters serve to buffer both an excess and a lack of free sterols. In this review, the esterification process, the storage of steryl esters and their mobilization will be described. Several model organisms are discussed but the focus was set on mammals and the yeast Saccharomyces cerevisiae. The contribution of imbalanced cholesterol homeostasis to several human diseases, namely Wolman disease, cholesteryl ester storage disease, atherosclerosis and Alzheimer's disease, Niemann-Pick type C and Tangier disease is described.

Involvement of a putative substrate binding site in the biogenesis and assembly of phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) produces the largest amount of cellular phosphatidylethanolamine (PE). Psd1p is synthesized as a larger precursor on cytosolic ribosomes and then imported into mitochondria in a three-step processing event leading to the formation of an α-subunit and a ß-subunit. The α-subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding. Here, we present a molecular analysis of this consensus motif for the function of Psd1p by using Psd1p variants bearing either deletions or point mutations in this region. Our data show that mutations in this motif affect processing and stability of Psd1p, and consequently the enzyme's activity. Thus, we conclude that this consensus motif is essential for structural integrity and processing of Psd1p.

The effect of hypoxia on the lipidome of recombinant Pichia pastoris.

BACKGROUND: Cultivation 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. RESULTS: To 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. CONCLUSIONS: Our 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.

Cell biology, physiology and enzymology of phosphatidylserine decarboxylase.

Phosphatidylethanolamine is one of the most abundant phospholipids whose major amounts are formed by phosphatidylserine decarboxylases (PSD). Here we provide a comprehensive description of different types of PSDs in the different kingdoms of life. In eukaryotes, type I PSDs are mitochondrial enzymes, whereas other PSDs are localized to other cellular compartments. We describe the role of mitochondrial Psd1 proteins, their function, enzymology, biogenesis, assembly into mitochondria and their contribution to phospholipid homeostasis in much detail. We also discuss briefly the cellular physiology and the enzymology of Psd2. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria.

Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.

Regulation of the yeast triacylglycerol lipases Tgl4p and Tgl5p by the presence/absence of nonpolar lipids.

Tgl3p, Tgl4p, and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae Recently we demonstrated that properties of Tgl3p are regulated by the formation of nonpolar lipids. The present study extends these investigations to the two other yeast triacylglycerol lipases, Tgl4p and Tgl5p. We show that Tgl4p and Tgl5p, which are localized to lipid droplets in wild type, are partially retained in the endoplasmic reticulum in cells lacking triacylglycerols and localize exclusively to the endoplasmic reticulum in a mutant devoid of lipid droplets. In cells lacking steryl esters, the subcellular distribution of Tgl4p and Tgl5p is unaffected, but Tgl5p becomes unstable, whereas the stability of Tgl4p increases. In cells lacking nonpolar lipids, Tgl4p and Tgl5p lose their lipolytic activity but retain their side activity as lysophospholipid acyltransferases. To investigate the regulatory network of yeast triacylglycerol lipases in more detail, we also examined properties of Tgl3p, Tgl4p, and Tgl5p, respectively, in the absence of the other lipases. Surprisingly, lack of two lipases did not affect expression, localization, and stability of the remaining Tgl protein. These results suggest that Tgl3p, Tgl4p, and Tgl5p, although they exhibit similar functions, act as independent entities.

A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.

Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

A Yeast Mutant Deleted of GPH1 Bears Defects in Lipid Metabolism.

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: 10.1371/journal.pone.0077380). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

Systems-level organization of yeast methylotrophic lifestyle.

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology.

Phosphatidylcholine affects the role of the sorting and assembly machinery in the biogenesis of mitochondrial ß-barrel proteins.

Two protein translocases drive the import of ß-barrel precursor proteins into the mitochondrial outer membrane: The translocase of the outer membrane (TOM complex) promotes transport of the precursor to the intermembrane space, whereas the sorting and assembly machinery (SAM complex) mediates subsequent folding of the ß-barrel and its integration into the target membrane. The non-bilayer-forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) are required for the biogenesis of ß-barrel proteins. Whether bilayer-forming phospholipids such as phosphatidylcholine (PC), the most abundant phospholipid of the mitochondrial outer membrane, play a role in the import of ß-barrel precursors is unclear. In this study, we show that PC is required for stability and function of the SAM complex during the biogenesis of ß-barrel proteins. PC further promotes the SAM-dependent assembly of the TOM complex, indicating a general role of PC for the function of the SAM complex. In contrast to PE-deficient mitochondria precursor accumulation at the TOM complex is not affected by depletion of PC. We conclude that PC and PE affect the function of distinct protein translocases in mitochondrial ß-barrel biogenesis.

Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.

Regulatory link between steryl ester formation and hydrolysis in the yeast Saccharomyces cerevisiae.

Steryl esters and triacylglycerols are the major storage lipids of the yeast Saccharomyces cerevisiae. Steryl esters are formed in the endoplasmic reticulum by the two acyl-CoA:sterol acyltransferases Are1p and Are2p, whereas steryl ester hydrolysis is catalyzed by the three steryl ester hydrolases Yeh1p, Yeh2p and Tgl1p. To shed light on the regulatory link between steryl ester formation and hydrolysis in the maintenance of cellular sterol and free fatty acid levels we employed yeast mutants which lacked the enzymes catalyzing the degradation of steryl esters. These studies revealed feedback regulation of steryl ester formation by steryl ester hydrolysis although in a Δtgl1Δyeh1Δyeh2 triple mutant the gene expression levels of ARE1 and ARE2 as well as protein levels and stability of Are1p and Are2p were not altered. Nevertheless, the capacity of the triple mutant to synthesize steryl esters was significantly reduced as shown by in vitro and in vivo labeling of lipids with [(14)C]oleic acid and [(14)C]acetate. Enzymatic analysis revealed that inhibition of steryl ester formation occurred at the enzyme level. As the amounts and the formation of sterols and fatty acids were also decreased in the triple mutant we concluded that defects in steryl ester hydrolysis also caused feedback inhibition on the formation of sterols and fatty acids which serve as precursors for steryl ester formation. In summary, this study demonstrates a regulatory link within the steryl ester metabolic network which contributes to non-polar lipid homeostasis in yeast cells.