Functional characterization of the phosphorelay protein Mpr1p from Schizosaccharomyces pombe

Histidine-containing phosphotransfer (HPt) proteins play an essential role in multistep histidine-aspartate phosphorelay signal transduction systems in prokaryotes and eukaryotes. The putative HPt protein in Schizosaccharomyces pombe, Mpr1p (also known as Spy1p), is a 295 amino acid protein that appears to be composed of more than one functional domain. The amino acid sequence of the N-terminal region of Mpr1p lacks homology to other known proteins, whereas the C-terminal domain is predicted to have structural similarity to the Ypd1p HPt protein from Saccharomyces cerevisiae. This study provides both in vitro and in vivo evidence that the C-terminal domain of Mpr1p indeed functions as an HPt protein in shuttling phosphoryl groups from one response regulator domain to another. Furthermore, we find that various deletions of the N-terminal region diminish both the phosphotransfer activity of Mpr1p and its affinity for response regulator domains, suggesting a possible role for the N-terminal domain in HPt-response regulator domain interactions.     

Characterization of AGT1 encoding a general α-glucoside transporter from Saccharomyces

Molecular genetic analysis is used to characterize the AGT1 gene encoding an α-glucoside transporter. AGT1 is found in many Saccharomyces cerevisiae laboratory strains and maps to a naturally occurring, partially functional allele of the MAL1 locus. Agt1p is a highly hydrophobic, postulated integral membrane protein. It is 57% identical to Mal61p, the maltose permease encoded at MAL6 , and is also a member of the 12 transmembrane domain superfamily of sugar transporters. Like Mal61p, Agt1p is a high-affinity, maltose/proton symporter, but Mal61p is capable of transporting only maltose and turanose, while Agt1p transports these two α-glucosides as well as several others including isomaltose, α-methylglucoside, maltotriose, palatinose, trehalose and melezitose. AGT1 expression is maltose inducible and induction is mediated by the Mal-activator. The sequence of the upstream region of AGT1 is identical to that of the maltose-inducible MAL61 gene over a 469 bp region containing the UAS_MAL but the 315 bp sequence immediately upstream of AGT1 shows no significant homology to the sequence immediately upstream of MAL61 . The evolutionary origin of the MAL1 allele to which AGT1 maps and the relationship of AGT1 to other α-glucoside fermentation genes is discussed.     

Functional expression and characterization of Arabidopsis ABCB,AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe

Heterologous expression systems based on tobacco BY‐2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway^®‐compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher ³H‐IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease‐deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration‐dependent reversal of ³H‐IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.     

Pgt1, a glutathione transporter from the fission yeast Schizosaccharomyces pombe

The Schizosaccharomyces pombe ORF, SPAC29B12.10c, a predicted member of the oligopeptide transporter (OPT) family, was identified as a gene encoding the S. pombe glutathione transporter ( Pgt1 ) by a genetic strategy that exploited the requirement of the cys1a Δ strain of S. pombe (which is defective in cysteine biosynthesis) for either cysteine or glutathione, for growth. Disruption of the ORF in the cys1a Δ strain led to an inability to grow on glutathione as a source of cysteine. Cloning and subsequent biochemical characterization of the ORF revealed that a high-affinity transporter for glutathione ( K _m=63 μM) that was found to be localized to the plasma membrane. The transporter was specific for glutathione, as significant inhibition in glutathione uptake could be observed only by either reduced or oxidized glutathione, or glutathione conjugates, but not by dipeptides or tripeptides. Furthermore, although glu–cys–gly, an analogue of glutathione (γ-glu–cys–gly), could be utilized as a sulphur source, the growth was not Pgt1 dependent. This further underlined the specificity of this transporter for glutathione. The strong repression of pgt1⁺ expression by cysteine suggested a role in scavenging glutathione from the extracellular environment for the maintenance of sulphur homeostasis in this yeast.     

Cloning and characterization of the kinesin-related protein,Krp1p,in Schizosaccharomyces pombe

Kinesin have been cloned in many organisms. They played important roles in the transport of cell organelles, polarized growth, and secretion. We report here the identification of a kinesin-related protein in Schizosaccharomyces pombe, which was named kinesin-related protein (Krplp). The primer sequences were driven from the highly conserved area of the kinesin genes in other organisms. We cloned kinesin genes from S. pombe using the PCR technique. Sequence analysis revealed that krp1+ has a 1,665 bp open-reading frame (ORF) that encoded a protein that consisted of 554 amino acids with a molecular weight of 61,900. It is homologous to the proteins that belong to the kinesin heavy chain (KHC) superfamily [GenBank accession No. AF156966 (genomic DNA) and AF247188 (mRNA)]. To characterize Krplp, the gene was disrupted and overexpressed in S. pombe. Cells that contained a krp1+ null allele were viable. Overexpression of Krp1p resulted in the inhibition of mitotic growth; cells became elongated, branched, and formed aberrant septa. To identify proteins that interact with Krplp, the yeast two-hybrid system was used. As a result, the novel protein, designated kinesin associated protein (Kap1p), was identified and showed structural homology to the proteins of the myosin family (GenBank accession No. AF351206). The data from the overexpression and two-hybrid study of Krplp may provide information that Krplp can have roles in cytokinesis with myosin.     

Abc1: a new ABC transporter from the fission yeast Schizosaccharomyces pombe

We have isolated the abc1 gene from the fission yeast Schizosaccharomyces pombe. Sequence analysis suggests that the Abc1 protein is a member of the ABC superfamily of transporters and is composed of two structurally homologous halves, each consisting of a hydrophobic region of six transmembrane domains and a hydrophilic region containing one ATP-binding site. The abc1 gene appears to be expressed under all growth conditions but gene disruption experiments indicate that it is not essential for growth. The sequence of the abc1 gene has been deposited in the EMBL data library under the Accession Number Y09354.     

Identification and characterization of a gene required for α1,2-mannose extension in the O-linked glycan synthesis pathway in Schizosaccharomyces pombe

The KTR α1,2-mannosyltransferase gene family of Saccharomyces cerevisiae is responsible not only for outer-chain modifications of N -linked oligosaccharides but also for elongation of O -linked mannose residues. To identify genes involved in the elongation step of O -linked oligosaccharide chains in Schizosaccharomyces pombe , we characterized six genes, omh1 ⁺ –omh6 ⁺, that share significant sequence similarity to the S. cerevisiae KTR family. Six deletion strains were constructed, each carrying a single disrupted omh allele. All strains were viable, indicating that none of the omh genes was essential. Heterologous expression of a chitinase from S. cerevisiae in the omh mutants revealed that O -glycosylation of chitinase had decreased in omh1 Δ cells, but not in the other mutants, indicating that the other omh genes do not appear to be required for O -glycan synthesis. Addition of the second α1,2-linked mannose residue was blocked in omh1 Δ cells. An Omh1–GFP fusion protein was found to be localized in the Golgi apparatus. These results indicate that Omh1p plays a major role in extending α1,2-linked mannose in the O -glycan pathway in S. pombe .     

A homologue of the ras-related CDC42 gene from Schizosaccharomyces pombe.

A cDNA was isolated from the fission yeast, Schizosaccharomyces pombe, using mixed oligodeoxyribonucleotides encoding part of the GTP-binding site of the ras superfamily. The encoded protein is the homologue of the budding yeast CDC42 gene product and the human proteins, CDC42Hs and G25K.     

Biochemical characterization of the structure-specific DNA-binding protein Cmb1 from Schizosaccharomyces pombe

Cmb1, a novel HMG box protein from Schizosaccharomyces pombe, has been characterized biochemically using glutaraldehyde cross-linking, gel-filtration and analytical ultracentrifugation. It was identified as a monomeric, non-spherical protein, with a tendency to aggregate in solution. Limited proteolysis with trypsin and chymotrypsin showed that the C-terminal HMG box was a compact, proteolytically stable domain and the N-terminal region of Cmb1 was relatively unstructured and more easily digested.As Cmb1 was previously identified as a potential mismatch-binding protein, the binding constants and stoichiometry for both homoduplex and heteroduplex DNA were determined using an IASys resonant mirror biosensor. Cmb1 indeed demonstrated a tighter association with mismatched DNA, especially with the C/Delta-mismatch. Expression constructs of Cmb1 were made to study the sections of the protein involved in DNA binding. Constructs with the N-terminal region absent revealed that the C-terminal HMG box was the primary DNA-binding region. The presence of the N-terminal region did, however, facilitate tighter binding to both homoduplex and heteroduplex DNA. The amino acid residues isoleucine 14 and leucine 39 were located as putative intercalating residues using structure guided homology modelling. The model templates were derived from two distinct HMG:DNA complexes: HMG-D bound to homoduplex DNA and HMG 1 bound to cisplatin DNA. Binding studies using the Cmb1 HMG box with point mutations in these residues showed that isoleucine 14 was important for the binding of Cmb1 to homoduplex DNA, but affected binding to mismatches to a lesser extent. In contrast, leucine 39 appeared to have a more significant function in binding to mismatched DNA.     

Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.     

Characterization of Thi9, a novel thiamine (Vitamin B1) transporter from Schizosaccharomyces pombe

Thiamine is an essential component of the human diet and thiamine diphosphate-dependent enzymes play an important role in carbohydrate metabolism in all living cells. Although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe can derive thiamine from biosynthesis, both are also able to take up thiamine from external sources, leading to the down-regulation of the enzymes involved in its formation. We have isolated the S. pombe thiamine transporter Thi9 by genetic complementation of mutants defective in thiamine biosynthesis and transport. Thi9 localizes to the S. pombe cell surface and works as a high-affinity proton/thiamine symporter. The uptake of thiamine was reduced in the presence of pyrithiamine, oxythiamine, amprolium, and the thiazole part of thiamine, indicating that these compounds are substrates of Thi9. In pyrithiamine-resistant mutants, a conserved glutamate residue close to the first of the 12 transmembrane domains is exchanged by a lysine and this causes aberrant localization of the protein. Thiamine uptake is significantly increased in thiamine-deficient medium and this is associated with an increase in thi9(+) mRNA and protein levels. Upon addition of thiamine, the thi9(+) mRNA becomes undetectable within minutes, whereas the Thi9 protein appears to be stable. The protein is distantly related to transporters for amino acids, gamma-aminobutyric acid and polyamines, and not to any of the known thiamine transporters. We also found that the pyridoxine transporter Bsu1 has a marked contribution to the thiamine uptake activity of S. pombe cells.     

Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe

We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h-1 while growth yield is lowered by 20% (from 1. 14 x 10(8) to 9.12 x 10(7) cells ml-1). Kinetic studies suggest that the effect on growth is mitochondrial in origin. In isolated mitochondria we found that the alternative oxidase actively competes with the cytochrome pathway for reducing equivalents and contributes up to 24% to the overall respiratory activity. Metabolic control analysis reveals that the alternative oxidase exerts a considerable degree of control (22%) on total electron flux. Furthermore, the negative control exerted by the alternative oxidase on the flux ratio of electrons through the cytochrome and alternative pathways is comparable with the positive control exerted on this flux-ratio by the cytochrome pathway. To our knowledge, this is the first paper to report a phenotypic effect because of plant alternative oxidase expression. We suggest that the effect on growth is the result of high engagement of the non-protonmotive alternative oxidase in yeast respiration that, consequently, lowers the efficiency of energy conservation and hence growth.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

Functional characterization of Candida albicans ABC transporter Cdr1p

In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).     

Dα3, a New Functional α Subunit of Nicotinic Acetylcholine Receptors from Drosophila

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are important excitatory neurotransmitter receptors in the insect CNS. We have isolated and characterized the gene and the cDNA of a new nAChR subunit from Drosophila . The predicted mature nAChR protein consists of 773 amino acid residues and has the structural features of an ACh-binding α subunit. It was therefore named Dα3, for D rosophila α -subunit 3 . The dα3 gene maps to the X chromosome at position 7E. The properties of the Dα3 protein were assessed by expression in Xenopus oocytes. Dα3 did not form functional receptors on its own or in combination with any Drosophila β-type nAChR subunit. Nondesensitizing ACh-evoked inward currents were observed when Dα3 was coexpressed with the chick β2 subunit. Half-maximal responses were at ∼0.15 µ M ACh with a Hill coefficient of ∼1.5. The snake venom component α-bungarotoxin (100 n M ) efficiently but reversibly blocked Dα3/β2 receptors, suggesting that Dα3 may be a component of one of the previously described two classes of toxin binding sites in the Drosophila CNS.     

Functional characterization of Dma1 and Dma2, the budding yeast homologues of Schizosaccharomyces pombe Dma1 and human Chfr

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Delta dma2Delta cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.     

Functional over-expression of the Stm1 protein,a G-protein-coupled receptor,in Schizosaccharomyces pombe

We report here the first functional over-expression of the Stm1 protein, a G-protein-coupled receptor with seven-trans-membrane spanning regions, in a homologous expression system without internal modification of the open reading frame of Stm1. The entire coding sequence, except for the termination codon followed by a C-terminal His6 tag, has been cloned into the pREP1 vector. The functionally active Stm1-His6 was over-expressed in Schizosaccharomyces pombe under the control of the nmt1 (no message in thiamine) promoter. The expression after induction was 120 times as much as that of control before induction and it gave 500 ng protein/2 × 10⁷cells.