A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform

The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure–function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [³H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.     

A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance

Sterols are major lipids in eukaryotes and differ in their specific structure between species. Both cholesterol and ergosterol can form liquid ordered domains in artificial membranes. We reasoned that substituting the main sterol ergosterol by cholesterol in yeast should permit domain formation and discriminate between physical and sterol structure-dependent functions. Using a cholesterol-producing yeast strain, we show that solute transporters for tryptophan and arginine are functional, whereas the export of weak organic acids via Pdr12p, a multi-drug resistance family member, is not. The latter reveals a sterol function that is probably dependent upon a precise sterol structure. We present a series of novel yeast strains with different sterol compositions as valuable tools to characterize sterol function and use them to refine the sterol requirements for Pdr12p. These strains will also be improved hosts for heterologous expression of sterol-dependent proteins and safe sources to obtain pure cholesterol and other sterols.     

Requirements for Recruitment of a G Protein-coupled Receptor to Clathrin-coated Pits in Budding Yeast

Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor Ste2p has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment of Ste2p to preexisting clathrin-coated pits (CCPs) as a key step regulated by receptor phosphorylation and subsequent ubiquitination upon ligand binding. The yeast casein kinase I homologue Yck2p directly phosphorylates six serine residues located in the C-terminal tail of Ste2p, and mutation of these serine residues to alanine significantly decreased recruitment of Ste2p to CCPs. We also found that the clathrin adaptors Ent1p, Ent2p, and Ede1p work cooperatively to recruit ubiquitinated Ste2p to CCPs. In addition, ubiquitination has a role in ligand-independent constitutive recruitment of Ste2p to CCPs, although this process is much slower than ligand-induced recruitment. These results suggest that ubiquitination of Ste2p is indispensable for recruiting Ste2p to CCPs in both ligand-dependent and ligand-independent endocytosis.     

GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles

The biophysical characterization of purified membrane proteins typically requires detergent mediated extraction from native lipid membrane environments. In the case of human G protein-coupled receptors (GPCRs), this process has been complicated by their conformational heterogeneity and the general lack of understanding the composition and interactions within the diverse human cellular membrane environment. Several successful GPCR structure determination efforts have shown that the addition of cholesterol analogs is often critical for maintaining protein stability. We have identified sterols that substantially increase the stability of the NOP receptor (ORL-1), a member of the opioid GPCR family, in a mixed micelle environment. Using dynamic light scattering and small-angle X-ray scattering, we have determined that the most thermal stabilizing sterol, cholesteryl hemisuccinate, induces the formation of a bicelle-like micelle architecture when mixed with dodecyl maltoside detergent. Together with mutagenesis studies and recent GPCR structures, our results provide indications that stabilization is attained through a combination of specific sterol binding to GPCRs and modulation of micelle morphology.     

Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.     

Analysis of ligand-receptor cross-linked fragments by mass spectrometry.

G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294.     

Influence of sterol structure on yeast plasma membrane properties

Fluorescence anisotropy measurements indicated that physical changes occured in the lipids of plasma membranes of yeast sterol mutants but not in the plasma membrane of an ergosterol wild-type. Parallel experiments with model membrane liposomes verified that the physical changes in lipids observed in the sterol mutants are dependent on the sterol present and not the phospholipid composition. In addition, the physical changes in lipids observed in liposomes derived from wild-type phospholipids were eliminated by addition of ergosterol but persisted in the presence of cholesterol, cholestanol, ergostanol, or sterols from the sterol mutants. No physical changes in lipids were observed, however, in plasma membranes from a sterol auxotroph, even when the auxotroph was grown on cholesterol or cholestanol. The lack of physical changes in lipids in the sterol auxotroph may reflect the ability of the auxotroph to modify its phospholipid composition with respect to its sterol composition. These results indicate that high specificity ‘sparking’ sterol is not required for the regulation of overall bulk lipid properties of the plasma membrane.     

Signaling of human frizzled receptors to the mating pathway in yeast

Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Galpha protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors.     

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p–[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.     

Peptide fragments as models to study the structure of a G-protein coupled receptor: the alpha-factor receptor of Saccharomyces cerevisiae

The alpha-factor tridecapeptide initiates mating in Saccharomyces cerevisiae upon interaction with Ste2p, its cognate G-protein coupled receptor (GPCR). This interaction is being used as a paradigm for understanding the structure and mechanism of activation of GPCRs by medium-sized peptides. In this article, the use of fragments of Ste2p to study its structure is reviewed. Methods of synthesis of peptides corresponding to both extramembranous and transmembrane domains of Ste2p are evaluated and problems that are encountered during synthesis and purification are described. The results from conformational analyses of the peptide fragments using fluorescence spectroscopy, CD, infrared spectroscopy, and NMR spectroscopy in organic-aqueous mixtures and in the presence of detergent micelles and lipid bilayers are critically reviewed. The data obtained to date provide biophysical evidence for the structure of different domains of Ste2p and indicate that peptides corresponding to these domains have unique biophysical tendencies. The studies carried out on Ste2p fragments indicate that valuable information concerning the structure of the intact receptor can be obtained by studying peptide fragments corresponding to domains of these polytopic integral membrane proteins.     

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.     

Multiple functions of sterols in yeast endocytosis

Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergDelta mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2Deltaerg6Delta and erg3Deltaerg6Delta cells exhibit a strong internalization defect of the alpha-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligand-receptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. ergDelta cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment.     

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.     

Anaerobiosis induces complex changes in sterol esterification pattern in the yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen. We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis. Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells. Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol). Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen. Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells.     

An improved bioluminescence‐based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor

The goal of this work was to improve the bioluminescence‐based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast‐expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G‐protein α‐subunit (Gpa1p) with the olfactory‐specific Gα_olf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence‐based odor‐sensing system using OR‐expressing yeast. Biotechnol. Bioeng. 2012; 109: 3143–3151. © 2012 Wiley Periodicals, Inc.     

Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast

The proton pumping H(+)-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the Saccharomyces cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Delta mutant that affects the synthesis of the ceramide-bound C26 very long chain fatty acid (Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. (2002) Mol. Biol. Cell 13, 4414-4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the substitution of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis or its association with detergent-resistant membrane domains (lipid "rafts"). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long chain fatty acid results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.     

Evidence for similarities and differences in the biosynthesis of fungal sterols

The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.     

Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane

It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multivesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.     

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

We present an example of expression and purification of a biologically active G-protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR alpha-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5% n-dodecyl maltoside (DM). Approximately 120 microg of purified alpha-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (K(d)) of the purified alpha-factor receptor in DM micelles was 28 nM as compared to K(d)=12.7 nM for Ste2p in cell membranes, and approximately 40% of the purified receptor was correctly folded as judged by ligand saturation binding. About 50% of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the alpha-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.