Homologous Expression System Research Articles

Identification of a carboxylesterase expressed in protoplasts using fluorescence‐activated cell sorting

A method was developed to identify plant carboxylesterases using a homologous expression system with the capacity for high-throughput screening based on fluorescence-activated cell sorting (FACS). Protoplasts of Arabidopsis thaliana were prepared and transfected with a mutated (Cys59Ser) Arabidopsis S-formylglutathione hydrolase (atsfghm), which encoded a carboxylesterase highly active in the hydrolysis of the vital marker methylumbelliferyl acetate (MUA) to the fluorophore methylumbelliferone (MU). Unlike all other Arabidopsis carboxylesterases studied to date, AtSFGH and its more stable mutant variant AtSFGHm are insensitive to inhibition by organophosphate insecticides, such as paraoxon. By making use of the combined traits of a high carboxylesterase activity towards MUA and a lack of sensitivity to paraoxon, FACS was employed to selectively collect catalytically active atsfghm-transformed protoplasts. A population of 400,000 protoplasts containing 8000 sfghm transformants was treated with paraoxon to inhibit endogenous esterase activity and then fed with MUA. Fluorescent cells expressing the AtSFGHm enzyme were then collected by FACS, and the presence of the respective transgene was confirmed by polymerase chain reaction, with 9.6% of the transformants recovered. We suggest that the use of FACS to identify other carboxylesterases which can be catalytically determined using plant cell fluorescence-based assays could be a powerful method for the high-throughput screening of new enzymes, especially those which do not express well in microbial hosts.

Molecular dissection and expression of the LdK39 kinesin in the human pathogen, Leishmania donovani

In this study we show for the first time the intracellular distribution of a K39 kinesin homologue in Leishmania donovani, a medically important parasite of humans. Further, we demonstrated that this motor protein is expressed in both the insect and mammalian developmental forms (i.e. promastigote and amastigotes) of this organism. Moreover, in both of these parasite developmental stages, immunofluorescence indicated that the LdK39 kinesin accumulated at anterior and posterior cell poles and that it displayed a peripheral localization consistent with the cortical cytoskeleton. Using a molecular approach, we identified, cloned and characterized the first complete open reading frame for the gene (LdK39) encoding this large (> 358 kDa) motor protein in L. donovani. Based on these observations, we subsequently used a homologous episomal expression system to dissect and express the functional domains that constitute the native molecule. Cell fractionation experiments demonstrated that LdK39 was soluble and that it bound to detergent-extracted cytoskeletons of these parasites in an ATP-dependent manner. The cumulative results of these experiments are consistent with LdK39 functioning as an ATP-dependent kinesin which binds to and travels along the cortical cytoskeleton of this important human pathogen.

Exclusive overproduction of recombinant versatile peroxidase MnP2 by genetically modified white rot fungus, Pleurotus ostreatus

By combining a homologous recombinant gene expression system and optimization of the culture conditions, hyper overproduction of Pleurtous ostreatus MnP2 was achieved. Genetically modified P. ostreatus strains with the recombinant mnp2 sequence under the control of sdi1 expression signals, were subjected to agitated culture using media supplemented with wheat bran or its hot-water extract. The best result, whereby 7300 U/l of MnP was produced by a recombinant strain TM2-18, indicated that more than 30-fold overproduction of the recombinant MnP2 compared to the previous result was achieved. On the other hand, no MnP activity was detected for the wild-type strain under the same conditions. Accumulation of the recombinant, but not endogenous, mnp2 transcripts was demonstrated in reverse-transcription PCR experiments. These results indicated that the recombinant MnP2 was exclusively expressed by the recombinant strain. Purified recombinant MnP2 showed almost identical properties to native MnP2 in electrophoresis, spectroscopic and kinetic analyses, including determination of K(m) and V(max) values for Mn(II), H(2)O(2) and veratryl alcohol. Moreover, the recombinant MnP2 directly oxidized a high-molecularweight substrate RNase A in the absence of redox mediators, as does native MnP2. The homologous overproduction system will provide a plat form for exclusive production of mutant or variant peroxidases with a desired property in basidiomycete.

Molecular Characterization, Expression, and in Vivo Analysis of LmexCht1: THE CHITINASE OF THE HUMAN PATHOGEN, LEISHMANIA MEXICANA

Chitinases have been implicated to be of importance in the life cycle development and transmission of a variety of parasitic organisms. Using a molecular approach, we identified and characterized the structure of a single copy LmexCht1-chitinase gene from the primitive trypanosomatid pathogen of humans, Leishmania mexicana. The LmexCht1 encodes an approximately 50 kDa protein, with well conserved substrate binding and catalytic domains characteristic of members of the chitinase-18 protein family. Further, we showed that LmexCht1 mRNA is constitutively expressed by both the insect vector (i.e. promastigote) and mammalian (i.e. amastigote) life cycle developmental forms of this protozoan parasite. Interestingly, however, amastigotes were found to secrete/release approximately >2-4-fold higher levels of chitinase activity during their growth in vitro than promastigotes. Moreover, a homologous episomal expression system was devised and used to express an epitope-tagged LmexCht1 chimeric construct in these parasites. Expression of the LmexCht1 chimera was verified in these transfectants by reverse transcription-PCR, Western blots, and indirect immunofluorescence analyses. Further, results of coupled immunoprecipitation/enzyme activity experiments demonstrated that the LmexCht1 chimeric protein was secreted/released by these transfected L. mexicana parasites and that it possessed functional chitinase enzyme activity. Such transfectants were also evaluated for their infectivity both in human macrophages in vitro and in two different strains of mice. Results of those experiments demonstrated that the LmexCht1 transfectants survived significantly better in human macrophages and also produced significantly larger lesions in mice than control parasites. Taken together, our results indicate that the LmexCht1-chimera afforded a definitive survival advantage to the parasite within these mammalian hosts. Thus, the LmexCht1 could potentially represent a new virulence determinant in the mammalian phase of this important human pathogen.

Molecular evidence that the rate‐limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.

A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Cytochrome ba 3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/ T. thermophilus shuttle vector. The ba 3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba 3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2 L) with typical yields of ∼2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba 3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c 552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba 3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses ∼5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 Å resolution electron-density map will be discussed elsewhere.

Interaction between the H2 sensor HupUV and the histidine kinase HupT controls HupSL hydrogenase synthesis in Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by H(2) through a signal transduction cascade comprising three proteins: the H(2)-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His(6))-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a His(6)-HupUHupV complex present in dimeric and tetrameric forms, both of which had H(2) uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, H(2) affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H(2) regulation.

Functional Defects in Six Ryanodine Receptor Isoform-1 (RyR1) Mutations Associated with Malignant Hyperthermia and Their Impact on Skeletal Excitation-Contraction Coupling

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MHRyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WTRyR1, six of the most common human MHRyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WTRyR1. The hypersensitivity of MH myotubes extended to K+ depolarization. MH myotubes responded to direct channel activators with maximum Ca2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of MHRyR1s were directly analyzed using [3H]ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (<or=100 nm), and all were significantly more responsive to caffeine than WTRyR1 at Ca2+ concentrations that approximate those in resting myotubes. All seven mutations had diminished sensitivity to inhibition by Ca2+ and Mg2+. Using a homologous expression system, our study demonstrates for the first time that these 7 MH mutations are all both necessary and sufficient to induce MH-related phenotypes. Decreased sensitivity to Ca2+ and Mg2+ inhibition and inability of MHRyR1s to be fully inactivated at [Ca2+]i typical of normal myotubes at rest are key defects that contribute to the initiation of MH episodes.

Role of the flavin domain residues, His689 and Asn732, in the catalytic mechanism of cellobiose dehydrogenase from phanerochaete chrysosporium.

Cellobiose dehydrogenase is an extracellular flavocytochrome, which catalyzes the oxidation of cellobiose and other soluble oligosaccharides to their respective lactones, while reducing various one- and two-electron acceptors. Two residues at the active site of the flavin domain, His689 and Asn732, have been proposed to play critical roles in the oxidation of the substrate. To test these proposals, each residue was substituted with either a Gln, Asn, Glu, Asp, Val, Ala, and/or a His residue by site-directed mutagenesis, using a homologous expression system previously developed in our laboratory. This enabled an examination of the functional, stereochemical, and electrostatic constraints for binding and oxidation of the substrate. The steady-state kinetic parameters for the variant proteins were compared using cellobiose and its epimer, lactose, as the substrates. The H689 variants all exhibit >1000-fold lower k(cat) values, while the K(m) values for both substrates in these variants are similar to that of the wild-type enzyme. This supports the proposed role of this His residue as a general base in catalysis. The N732 variants exhibit a range of kinetic parameters: the k(cat) values for oxidation are 5-4000-fold lower than that for the wild-type enzyme, while the K(m) values vary between similar to and 60-fold higher than that for the wild-type. The difference in binding energy between cellobiose and lactose was calculated using the relationship delta(delta G) = -RT ln[(k(cat)/K(m))(lactose)/(k(cat)/K(m))(cellobiose)]. This calculation for the wild-type enzyme suggests that lactose binds considerably more weakly than cellobiose (7.2 kJ/mol difference), which corresponds to one extra (cumulative) hydrogen bond for cellobiose over lactose. Mutations at Asn732 result in a further weakening of lactose binding over cellobiose (2-4 kJ/mol difference). The results support a role for Asn732 in the binding of the substrate.

Outer sphere mutagenesis of Lactobacillus plantarum manganese catalase disrupts the cluster core. Mechanistic implications.

X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure9, 725-738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a micro 1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an 'open' form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km for H2O2 ( approximately 1.4 m) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.

Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O(2)- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (alphabetagamma)(2) complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the alpha subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

Molecular Dissection of the Functional Domains of a Unique, Tartrate-resistant, Surface Membrane Acid Phosphatase in the Primitive Human Pathogen Leishmania donovani

The primitive trypanosomatid pathogen of humans, Leishmania donovani, constitutively expresses a unique externally oriented, tartrate-resistant, acid phosphatase on its surface membrane. This is of interest because these organisms are obligate intracellular protozoan parasites that reside and multiply within the hydrolytic milieu of mammalian macrophage phago-lysosomes. Here we report the identification of the gene encoding this novel L. donovani enzyme. In addition, we characterized its structure, demonstrated its constitutive expression in both parasite developmental forms, and determined the cell surface membrane localization of its translated protein product. Further, we used a variety of green fluorescent protein chimeric constructs as reporters in a homologous leishmanial expression system to dissect the functional domains of this unique, tartrate-resistant, surface membrane enzyme.

Generating new marine cell lines and transgenic species--conference summary.

Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment. Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others. Much still remains to be learned from these animals, and there is a growing need for new marine models. Models for expression of marine animal genes have been limited to heterologous expression systems. While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible. This has become particularly important with the development of functional genomics in marine models. These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology.

Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system

Background & Aims:Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult. To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae. The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system. Methods:The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms 1219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH1Δ9/10) were cloned. On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay. Methods:Transfection was successful for all hMLH1 constructs. As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts. The V185G, G244D, and Δ9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the 1219V, R265H, R265C, R217C, and V326A mutations were MMR competent. Conclusions:These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products in a homologous expression system.

Type 1 and Type 3 Ryanodine Receptors Generate Different Ca 2+ Release Event Activity in Both Intact and Permeabilized Myotubes

In this investigation we use a “dyspedic” myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca 2+ release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca 2+ release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca 2+], as well as occasional spontaneous, propagating Ca 2+ release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K + depolarization. Expression of RyR1 restored depolarization-induced global Ca 2+ release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca 2+ release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K + depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted “Ca 2+ spark”-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca 2+ release behavior of RyR1 and RyR3 in this homologous expression system.

Dissection of the Functional Domains of theLeishmania Surface Membrane 3′-Nucleotidase/Nuclease, a Unique Member of the Class I Nuclease Family

Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family.

The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum.

Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes. Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far. Recently, the gene for an extremely halophilic beta-galactosidase (bgaH) from Haloferax alicantei has become available. After transformation of Halobacterium salinarum with a vector-carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate. Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific beta-galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT-PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea.

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae: PREPARATION AND UTILIZATION OF A GIANT CELL CONTAINING A GIANT VACUOLE

A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 micrometer in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/micrometer(2)) toward the inside of the vacuole. Bafilomycin A(1,) a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K(i) = 10 nM). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Deltavma1::TRP)). The K(m) value for the ATP dose response of the current was 159 microM and the H(+)/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 +/- 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

Functional reconstitution and characterization of AqpZ, the E. coli water channel protein

Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of ∼2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1 % SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1 % SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability ( p f ⩾ 10 × 10 −14 cm 3 s −1 subunit −1) and low Arrhenius activation energy ( E a = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.

Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.

An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b.