RESUMEN
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.
Asunto(s)
Proteínas Fúngicas/química , Redes y Vías Metabólicas , Proteoma , Saccharomycetales/genética , Biotecnología , Proteínas Fúngicas/genética , Metanol/metabolismo , Peroxisomas/metabolismo , Saccharomycetales/química , Fracciones SubcelularesRESUMEN
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.
Asunto(s)
Gotas Lipídicas/química , Fosfatidilgliceroles/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Fosfolipasas de Tipo C/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Metabolismo de los Lípidos , Mitocondrias/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Lípidos de la Membrana/química , Estrés Oxidativo , Vino/microbiología , Levaduras/fisiología , Ácidos Grasos Insaturados/análisis , Fermentación , Peróxido de Hidrógeno/farmacología , Lípidos de la Membrana/metabolismo , Metschnikowia/efectos de los fármacos , Metschnikowia/fisiología , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Esteroles/análisis , Esteroles/metabolismo , Torulaspora/efectos de los fármacos , Torulaspora/fisiología , Vino/análisis , Levaduras/efectos de los fármacosRESUMEN
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.
Asunto(s)
Carboxiliasas/química , Carboxiliasas/aislamiento & purificación , Membranas Mitocondriales/química , Proteínas Mitocondriales/química , Proteínas Mitocondriales/aislamiento & purificación , Señales de Clasificación de Proteína , Saccharomyces cerevisiae/química , Carboxiliasas/genética , Carboxiliasas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Dominios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosfatidilcolinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Carboxiliasas/metabolismo , Secuencia de Aminoácidos , Animales , Homeostasis/fisiología , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolípidos/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Aterosclerosis/metabolismo , Colesterol/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Tangier/metabolismo , Enfermedad de Wolman/metabolismo , Enfermedad de Alzheimer/genética , Animales , Aterosclerosis/genética , Colesterol/genética , Esterificación , Humanos , Enfermedad de Niemann-Pick Tipo C/genética , Saccharomyces cerevisiae/metabolismo , Enfermedad de Tangier/genética , Enfermedad de Wolman/genética , Enfermedad de Wolman/patologíaRESUMEN
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.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Retículo Endoplásmico/metabolismo , Gotas Lipídicas/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol Esterasa/metabolismo , Hidrolasas de Éster Carboxílico/genética , Retículo Endoplásmico/genética , Lípidos de la Membrana/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esterol Esterasa/genéticaRESUMEN
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.
Asunto(s)
Sitios de Unión/genética , Carboxiliasas/genética , Carboxiliasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Secuencias de Aminoácidos/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Mutación Puntual/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Metabolismo de los Lípidos/genética , Lípidos de la Membrana/metabolismo , Pichia/metabolismo , Respuesta de Proteína Desplegada , Ergosterol/biosíntesis , Fluconazol/farmacología , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Glucólisis , Lípidos de la Membrana/química , Oxígeno/metabolismo , Pichia/efectos de los fármacos , Pichia/genética , Pichia/crecimiento & desarrollo , Regiones Promotoras Genéticas , Transporte de Proteínas , Proteómica , Proteínas Recombinantes/metabolismo , Esfingolípidos/química , Esteroles/químicaRESUMEN
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.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriales/genética , Fosfatidilcolinas/genética , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Ésteres/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol Esterasa/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Estabilidad de Enzimas , Ácidos Grasos/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Hidrólisis , Metabolismo de los Lípidos/genética , Organismos Modificados Genéticamente , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol Esterasa/genética , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Glicerol/metabolismo , Metanol/metabolismo , Pichia/genética , Proteínas Fúngicas/metabolismo , Pichia/metabolismoRESUMEN
Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.
Asunto(s)
Citosol/enzimología , Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , Lipasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Retículo Endoplásmico/genética , Estabilidad de Enzimas , Lipasa/genética , Estructura Terciaria de Proteína , Proteolisis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Pichia/metabolismo , Ergosterol/metabolismo , Ácidos Grasos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , Esteroles/metabolismoRESUMEN
Tgl3p, Tgl4p and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae catalyzing degradation of triacylglycerols stored in lipid droplets. Previous results from our laboratory (Athenstaedt and Daum, 2005, J. Biol. Chem. 280, 37301-37309) demonstrated that a yeast strain lacking all three triacylglycerol lipases accumulates not only triacylglycerols at high amount, but also steryl esters. Here we show a metabolic link between synthesis and mobilization of non-polar lipids. In particular, we demonstrate that a block in tri-acylglycerol degradation in a tgl3∆tgl4∆tgl5∆ triple mutant lacking all major triacylglycerol lipases causes marked changes in non-polar lipid synthesis. Under these conditions formation of triacylglycerols is reduced, whereas steryl ester synthesis is enhanced as shown by quantification of non-polar lipids, in vivo labeling of lipids using [(14)C]oleic acid and [(14)C]acetic acid as precursors, and enzyme analyses in vitro. In summary, this study demonstrates that triacylglycerol metabolism and steryl ester metabolism are linked processes. The importance of balanced storage and degradation of these components for lipid homeostasis in the yeast is highlighted.
RESUMEN
The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.
Asunto(s)
Proteínas Fúngicas/análisis , Lípidos/análisis , Microsomas/química , Pichia/ultraestructura , Proteoma/análisis , Glicerofosfolípidos/análisis , Pichia/química , Control de Calidad , Esfingolípidos/análisisRESUMEN
Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections.
Asunto(s)
Antígenos Bacterianos/análisis , Vacunas Bacterianas/aislamiento & purificación , Exosomas/química , Haemophilus influenzae/química , Proteoma/análisis , Animales , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/química , Vacunas Bacterianas/inmunología , Exosomas/ultraestructura , Haemophilus influenzae/ultraestructura , Lípidos/análisis , Lipopolisacáridos/análisis , Ratones Endogámicos BALB C , Proteoma/inmunologíaRESUMEN
Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501-510). Here, we describe the effects of the presence/absence of triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.
Asunto(s)
Retículo Endoplásmico/enzimología , Lipasa/metabolismo , Metabolismo de los Lípidos/fisiología , Lípidos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Retículo Endoplásmico/genética , Estabilidad de Enzimas/fisiología , Eliminación de Gen , Genes Fúngicos , Lipasa/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of ß-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of ß-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of ß-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.