Thierry Magnin - Academia.edu (original) (raw)

Papers by Thierry Magnin

Philosophical Transactions of the Royal Society B, Apr 29, 1996

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation o... more Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (σ), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called σ F , the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit σ F . In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and σ F is set free. Release of σ F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/A DP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by σ F . First it regulates σ F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Protein Expression and Purification, Mar 1, 2009

major therapeutic importance. Structure determination of G protein-coupled receptors and other ap... more major therapeutic importance. Structure determination of G protein-coupled receptors and other appli-27 cations require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs 28 fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an 29 efficient method for their rapid purification that relies on the capture of these receptors with streptavidin 30 immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. 31 This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to 32 high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies 33 when large quantities of purified GPCRs are needed.

Plant Physiology, 1995

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After so... more The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95,66,56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis fhaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [y3*P]ATP in vitro. l h e complete loss of radioactivity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

Comptes rendus des séances de la Société de biologie et de ses filiales, 1992

Journal of Bacteriology, 1997

sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is ... more sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP. The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadi...

Genes & Development, 1994

Genetic experiments have suggested that σF, the first compartment-specific transcription factor i... more Genetic experiments have suggested that σF, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-σ factor SpoIIAB and an anti-anti-σ factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of σF-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA–phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA–phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-σF activity, whereas S...

Journal of Bacteriology, 1996

We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions... more We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB. Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP. SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP. SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP. Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis. SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P. Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in...

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After so... more The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95,66,56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis fhaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [y3*P]ATP in vitro. l h e complete loss of radioactivity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

Protein Science, 2006

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the... more We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Protein Expression and Purification, 2008

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins... more A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.

Protein Expression and Purification, 2009

a b s t r a c t G protein-coupled receptors (GPCRs) constitute the largest family of membrane rec... more a b s t r a c t G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

Plant Science, 1991

ABSTRACT Sealed tonoplast vesicles were prepared from vacuoles of Acer pseudoplatanus cells. The ... more ABSTRACT Sealed tonoplast vesicles were prepared from vacuoles of Acer pseudoplatanus cells. The rate of contamination by plasmalemma never exceeded 3%. The vesicles of tonoplast exhibited ATP- and PPi-dependent proton translocation activity. The properties of ATP-dependent H+-transport were studied and compared to those of ATP hydrolysis previously described (A. Pugin et al., in: B. Marin (Ed.), Plant Vacuoles, NATO ASI Series, Serie A, Vol. 134, Plenum Press, 1987, pp. 135–141). H+-transport and enzymatic properties of the tonoplast ATPase were well correlated. The ATP-dependent H+-translocation into the vesicles had an optimum around pH 7.3; MgATP2− was preferentially used as a substrate with an apparent Km of 153 μM. ATP-dependent H+ transport was stimulated by anions. This effect was shown to be partly due to the ability of permeant anions to dissipate the electrical potential Δψ. When Δψ was collapsed, H+-ATPase activity was stimulated by Cl− and inhibited by NO3−. The use of three pH probes: acridine orange (AO), 9-amino-6-chloro-2-methoxyacridine (ACMA) and quinacrine confirmed the limits of acridine orange for the measurement of pH gradients in the presence of NO3−. Various chemicals were potent inhibitors of the tonoplast H+-ATPase, the sulfhydryl reagents N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), mersalyl, the lipid soluble carbodiimide N,N′-dicyclohexylcarbodiimide (DCCD), the synthetic estrogen diethylstilbestrol (DES) and vanadate which is often reported as a specific inhibitor of the plasmalemma ATPase. The inefficiency of vanadate on the tonoplastic PPi-dependent H+ transport indicated that the inhibition of the ATP-dependent H+-pump resulted from an effect on the enzyme. The sensitivity of the tonoplastic H+-ATPase to vanadate is discussed. We suggest that there are V-type ATPases which could operate via a phosphoenzyme intermediate.

Plant Science, 1994

ABSTRACT Tonoplast H+-ATPase and H+-pyrophosphatase (H+-PPase) were previously characterized in A... more ABSTRACT Tonoplast H+-ATPase and H+-pyrophosphatase (H+-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H+-transports were also additive. The H+ -PPase inhibition did not change ATP-dependent H+-transport but H+-ATPase inhibition inhibited the PPi dependent H+-transport. Because H+-PPase was reported to transport H+ and K+ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H+-ATPase activity could modify the H+/K+ stoichiometry for the benefit of K+-transport.

Philosophical Transactions of the Royal Society B: Biological Sciences, 1996

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation o... more Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Molecular Microbiology, 1996

Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Baci... more Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis. Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated. Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein/protein complex. The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer. Its dissociation constant was 13 mu M. Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA. Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.

Journal of Structural and Functional Genomics, 2007

Production of recombinant receptors has been one of the major bottlenecks in structural biology o... more Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Glycobiology, 2003

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In f... more Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPIanchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C 24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Mana1-3Mana1-2Mana1-2Mana1-6Mana1-4GlcN.

Genes to Cells, 1996

Background: Differential gene expression during sporulation in the prespore and mother cell of Ba... more Background: Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (j) factors. The first j factor activated is j F , which directs gene expression specifically in the prespore compartment. Release of j F activity is tightly controlled through a series of complex interactions involving an anti-j factor, SpoIIAB, an anti-anti-j factor SpoIIAA and a phosphoprotein phosphatase SpoIIE. In vitro studies have shown that SpoIIAB binds to j F , preventing transcription of the j F regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it. However, nonphosphorylated SpoIIAA can displace j F from SpoIIAB. The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P.

Philosophical Transactions of the Royal Society B, Apr 29, 1996

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation o... more Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (σ), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called σ F , the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit σ F . In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and σ F is set free. Release of σ F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/A DP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by σ F . First it regulates σ F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Protein Expression and Purification, Mar 1, 2009

major therapeutic importance. Structure determination of G protein-coupled receptors and other ap... more major therapeutic importance. Structure determination of G protein-coupled receptors and other appli-27 cations require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs 28 fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an 29 efficient method for their rapid purification that relies on the capture of these receptors with streptavidin 30 immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. 31 This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to 32 high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies 33 when large quantities of purified GPCRs are needed.

Plant Physiology, 1995

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After so... more The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95,66,56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis fhaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [y3*P]ATP in vitro. l h e complete loss of radioactivity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

Comptes rendus des séances de la Société de biologie et de ses filiales, 1992

Journal of Bacteriology, 1997

sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is ... more sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP. The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadi...

Genes & Development, 1994

Genetic experiments have suggested that σF, the first compartment-specific transcription factor i... more Genetic experiments have suggested that σF, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-σ factor SpoIIAB and an anti-anti-σ factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of σF-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA–phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA–phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-σF activity, whereas S...

Journal of Bacteriology, 1996

We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions... more We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB. Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP. SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP. SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP. Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis. SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P. Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in...

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After so... more The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95,66,56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis fhaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [y3*P]ATP in vitro. l h e complete loss of radioactivity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

Protein Science, 2006

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the... more We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

Protein Expression and Purification, 2008

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins... more A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10mg of more than 90% pure TolAIII-Bcl-x(L)DeltaC and TolAIII-Bcl-2(2)DeltaC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing > 12 mg of Bcl-x(L)DeltaC or > 6 mg of Bcl-2(2)DeltaC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x(L)DeltaC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)DeltaC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an alpha-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x(L) proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x(L)DeltaC and Bcl-2(2)DeltaC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.

Protein Expression and Purification, 2009

a b s t r a c t G protein-coupled receptors (GPCRs) constitute the largest family of membrane rec... more a b s t r a c t G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.

Plant Science, 1991

ABSTRACT Sealed tonoplast vesicles were prepared from vacuoles of Acer pseudoplatanus cells. The ... more ABSTRACT Sealed tonoplast vesicles were prepared from vacuoles of Acer pseudoplatanus cells. The rate of contamination by plasmalemma never exceeded 3%. The vesicles of tonoplast exhibited ATP- and PPi-dependent proton translocation activity. The properties of ATP-dependent H+-transport were studied and compared to those of ATP hydrolysis previously described (A. Pugin et al., in: B. Marin (Ed.), Plant Vacuoles, NATO ASI Series, Serie A, Vol. 134, Plenum Press, 1987, pp. 135–141). H+-transport and enzymatic properties of the tonoplast ATPase were well correlated. The ATP-dependent H+-translocation into the vesicles had an optimum around pH 7.3; MgATP2− was preferentially used as a substrate with an apparent Km of 153 μM. ATP-dependent H+ transport was stimulated by anions. This effect was shown to be partly due to the ability of permeant anions to dissipate the electrical potential Δψ. When Δψ was collapsed, H+-ATPase activity was stimulated by Cl− and inhibited by NO3−. The use of three pH probes: acridine orange (AO), 9-amino-6-chloro-2-methoxyacridine (ACMA) and quinacrine confirmed the limits of acridine orange for the measurement of pH gradients in the presence of NO3−. Various chemicals were potent inhibitors of the tonoplast H+-ATPase, the sulfhydryl reagents N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), mersalyl, the lipid soluble carbodiimide N,N′-dicyclohexylcarbodiimide (DCCD), the synthetic estrogen diethylstilbestrol (DES) and vanadate which is often reported as a specific inhibitor of the plasmalemma ATPase. The inefficiency of vanadate on the tonoplastic PPi-dependent H+ transport indicated that the inhibition of the ATP-dependent H+-pump resulted from an effect on the enzyme. The sensitivity of the tonoplastic H+-ATPase to vanadate is discussed. We suggest that there are V-type ATPases which could operate via a phosphoenzyme intermediate.

Plant Science, 1994

ABSTRACT Tonoplast H+-ATPase and H+-pyrophosphatase (H+-PPase) were previously characterized in A... more ABSTRACT Tonoplast H+-ATPase and H+-pyrophosphatase (H+-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H+-transports were also additive. The H+ -PPase inhibition did not change ATP-dependent H+-transport but H+-ATPase inhibition inhibited the PPi dependent H+-transport. Because H+-PPase was reported to transport H+ and K+ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H+-ATPase activity could modify the H+/K+ stoichiometry for the benefit of K+-transport.

Philosophical Transactions of the Royal Society B: Biological Sciences, 1996

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation o... more Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Molecular Microbiology, 1996

Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Baci... more Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis. Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated. Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein/protein complex. The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer. Its dissociation constant was 13 mu M. Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA. Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.

Journal of Structural and Functional Genomics, 2007

Production of recombinant receptors has been one of the major bottlenecks in structural biology o... more Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Glycobiology, 2003

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In f... more Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPIanchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C 24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Mana1-3Mana1-2Mana1-2Mana1-6Mana1-4GlcN.

Genes to Cells, 1996

Background: Differential gene expression during sporulation in the prespore and mother cell of Ba... more Background: Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (j) factors. The first j factor activated is j F , which directs gene expression specifically in the prespore compartment. Release of j F activity is tightly controlled through a series of complex interactions involving an anti-j factor, SpoIIAB, an anti-anti-j factor SpoIIAA and a phosphoprotein phosphatase SpoIIE. In vitro studies have shown that SpoIIAB binds to j F , preventing transcription of the j F regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it. However, nonphosphorylated SpoIIAA can displace j F from SpoIIAB. The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P.