Genetic characterization of the secretory mutant MS-1 of Tetrahymena thermophila: vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation.

The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1 of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitutively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed. The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila.     

Genetic characterization of the secretory mutant MS-1 of Tetrahymena thermophila: Vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation

The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1, of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitu-tively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed. The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila. © 1992 Wiley-Liss, Inc.     

Mitochondrial Dehydrogenases in Different Taxa of Tetrahymena: Effect of Insulin

Mitochondrial dehydrogenase activity was measured in seven taxa of Tetrahymena (T. pyriformis G1, T. hegewishi, T. malaccensis, T. pigmentosa, T. shapiro, T. thermophila CU-399, T. thermophila MS-1). Enzyme activity was different in the taxa investigated. Insulin reduced enzyme activity in six of the seven taxa studied. The duration of activity reduction was relatively long (5–10 min.) in most of the cases, and in T. hegewishi this lasted up to the end of the measurements (30 min.). There was no interrelation between the basic dehydrogenase activity of the taxon and the effect of insulin. There was also no correlation between the degree of relationship (of the taxa) and the dehydrogenase profile after insulin treatment.     

Deficiency in lysosomal enzyme secretion is associated with upregulation of phosphatidylinositol 4-phosphate in tetrahymena

ABSTRACT. A variety of lower eukaryotes and certain mammalian cells are known to constitutively secrete lysosomal hydrolases. Recent studies in Tetrahymena have shown that phosphatidylinositol 3-phosphate regulates the proper secretion of lysosomal enzymes at the level of phagolysosome formation. We extend these findings by studying the secretion-deficient strain MS-1 of Tetrahymena thermophila , which possess phosphatidylinositol levels similar to wild type. However, steady-state levels of phosphatidylinositol 4-phosphate (PtdIns4P) were found to be doubled in this strain compared with wild type as shown by in vivo [³H]inositol labeling and high-performance liquid chromatography analysis. The increased PtdIns4P levels in MS-1 cells were unrelated to the upregulation of total phosphatidylinositol phosphate induced by hyperosmotic stress because this treatment resulted in a similar increase of PtdIns4P in MS-1 and wild-type cells. Hyperosmotic stress did not affect secretion in either of the two types of cells. On the other hand, under conditions of wortmannin-induced hypersecretion in wild-type cells, MS-1 cells developed a highly vacuolated phenotype while secretion was not induced. Importantly, comparative analysis of wild-type and MS-1 cells under wortmannin treatment showed that PtdIns4P levels were differentially regulated in the two strains. These results collectively suggest that PtdIns4P turnover in Tetrahymena is linked to lysosome secretion.     

Cytochemical investigation into the Ca-dependence of positive and negative hormonal imprinting in Tetrahymena

Insulin gives rise to positive imprinting in Tetrahymena pyriformis, but to negative imprinting in T. thermophila, as revealed by the respective increases and decreases in the insulin-binding capacity of these organisms observed during later interactions with this hormone. We found that changes in insulin-binding capacity exhibited parallelism with fluctuations of the levels of free, intracellular Ca2+ detectable by Quin-2 labeling. An exception was the second interaction of T. thermophila with insulin, which although showing a positive trend, produced a relatively small increase in the level of intracellular Ca2+. These observations suggest an interrelationship between hormone-binding capacity and the fluctuation of intracellular Ca2+ levels. Either hormone binding depends on the availability of Ca2+, or, alternatively, the latter depends on the binding capacity. Further studies are required to elucidate the true nature of this interdependence.     

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [³⁵S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH₄Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH₄Cl-treated cells. NH₄Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.     

Biosynthesis of secreted beta-hexosaminidase in Tetrahymena thermophila. A comparison of the wild type with a secretory mutant.

The synthesis and secretion of beta-hexosaminidase was studied in wild type and secretion-deficient Tetrahymena thermophila cells by metabolic labelling and immunoprecipitation. beta-Hexosaminidase is synthesized as a Mr 79,000 polypeptide which is within 10 min converted into a Mr 59,000 form. The Mr 59,000 polypeptide is further processed (within 20 min) into at least three major mature forms of Mr 58,000-54,000, which are almost quantitatively secreted into the culture medium within 1-2 h after their synthesis. Both precursor and mature forms contain asparagine-linked oligosaccharide chains which are cleavable by endoglucosaminidase F, but not by endoglucosaminidase H. Neither [32P]orthophosphate nor [35S]sulphate are incorporated into immunoprecipitable precursor and mature beta-hexosaminidases, suggesting the absence of a phosphorylated recognition marker. Biosynthesis and processing of beta-hexosaminidase is apparently unaltered in the secretory mutant MS-1; however the processed polypeptides remain cellular bound in the mutant, indicating that the mutation affects a late event in the secretion pathway of lysosomal enzymes.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

D-3 phosphoinositides of the ciliate Tetrahymena: characterization and study of their regulatory role in lysosomal enzyme secretion

Phosphatidylinositol 3-phosphate, PtdIns3P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns3,5P2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns3P and PtdIns3,5P2 were found to comprise 16% and 30-40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1-10 microM) resulted in the depletion of PtdIns3P and PtdIns3,5P2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes.     

Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis.

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 micrograms/ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 microgram/ml (P less than 0.01) but the staining intensity at 0 microgram/ml was not different from that at 3 micrograms/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo- mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.     

Mutations to the third cytoplasmic domain of the glucagon-like peptide 1 (GLP-1) receptor can functionally uncouple GLP-1-stimulated insulin secretion in HIT-T15 cells.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone with powerful antidiabetogenic effects that are thought to be mediated by adenylyl cyclase (AC). Recently, we generated two GLP-1 receptor mutant isoforms (IC3-1 and DM-1) that displayed efficient ligand binding and the ability to promote Ca2+ mobilization from intracellular stores but lacked the ability to couple to AC. In the present study, the wild-type rat GLP-1 receptor (WT-GLP-1 R) or the IC3-1 and DM-1 mutant forms were expressed for the first time in the insulin-producing HIT-T15 cells. Only cells expressing WT-GLP-1 R displayed dramatically elevated GLP-1-induced cAMP responses and elevated insulin secretion. The increase in GLP-1-stimulated secretion in cells expressing WT-GLP-1 R, however, was not accompanied by differences in glucose-stimulated insulin release. Prolonged exposure to GLP-1 (10 nM, 17 h), not only led to an increase in insulin secretion but also increased insulin mRNA levels, but only in cells expressing the WT-GLP-1 R and not the mutant isoforms. Electrophysiological analyses revealed that GLP-1 application enhanced L-type voltage-dependent Ca2+ channel (VDCC) currents > 2-fold and caused a positive shift in VDCC voltage-dependent inactivation in WT-GLP-1R cells only, not control or mutant (DM-1) cells. This action on the Ca2+ current was further enhanced by the VDCC agonist, BAYK8644, suggesting GLP-1 acts via a distinct mechanism dependent on cAMP. These studies demonstrate that the GLP-1 receptor efficiently couples to AC to stimulate insulin secretion and that receptors lacking critical residues in the proximal region of the third intracellular loop can effectively uncouple the receptor from cAMP production, VDCC activity, insulin secretion, and insulin biosynthesis.     

Taxon-dependence of receptor level cell-to-cell communication in Tetrahymena: possible explanation for the transmission of hormonal imprinting

Insulin treatment induced in Tetrahymena pyriformis a positive hormonal imprinting, and in Tetrahymena thermophila a negative imprinting, resulting in increased and decreased binding capacity, respectively, at re-exposure to the hormone. The imprinting, or the information associated with it, is transferred by the nutrient medium of the insulin-treated cells to those not treated. The issue of transfer depends on the nature of the receiver taxon, leading always to a positive imprinting in Tetrahymena pyriformis, and to a negative imprinting in Tetrahymena thermophila, regardless of the nature of the 'imprinted' transmitter taxon. The findings substantiate the transferability of hormonal imprinting by the nutrient medium at the unicellular level, the key role of the postreceptorial mechanism in determining the trend of imprinting and may explain the persistence of imprinting in the progeny generations.     

Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena.

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate+H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10(-4)M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.     

定向进化提高嗜热拟青霉J18耐热β-1,3-1,4-葡聚糖酶在酸性条件下的催化能力

应用定向进化技术提高了嗜热拟青霉Paecilomyces thermophila J18耐热β-1,3-1,4-葡聚糖酶(PtLic16A)在酸性条件下的催化能力.结合易错PCR和DNA改组的方法,构建了β-葡聚糖酶的突变体文库;利用刚果红染色法建立了阳性克隆的高通量筛选体系.筛选得到的突变酶PtLic 16AM1的反应最适pH由7.0变化至5.5,且保持了原有的耐热性和比酶活.突变酶的DNA序列中有4个点位发生突变,引发了4处氨基酸替换,分别是T58S、Y110N、G195E和D221G.结构模拟结果显示,发生突变的4个氨基酸位点中,Y110N位置靠近酶活性中心,而T58S、G195E和D221G则离酶活性中心较远,其中T58S、G195E可能对酶最适pH的变化起到了关键作用.     

Hydroxamate production by Aquaspirillum magnetotacticum.

Spent culture fluids from Aquaspirillum magnetotacticum MS-1 grown at high (20 microM) but not low (5 microM) iron concentration contained material yielding a positive hydroxamate test. Cells possessed six major outer membrane proteins. Three outer membrane proteins ranging from 72,000 to 85,000 daltons were coordinately produced at iron concentrations conducive to hydroxamate production. A 55,000-dalton iron-repressible outer membrane protein was also present in strain MS-1 cultured at low but not high ferric quinate concentration. Culture fluids from strain MS-1 which were hydroxamate positive augmented growth of a Salmonella typhimurium siderophore-deficient (enb-7) mutant in low-iron medium, suggesting a role of hydroxamate in uptake of iron by the cell.     

Effect of vanadate and ouabain on insulin binding and insulin imprinting in Tetrahymena.

Na-metavanadate and ouabain that act on Na+K(+)-ATPase had no influence on insulin binding to Tetrahymena immediately after treatment, but after 24 h considerably enhanced the binding capacity of generations of progeny. The increase in binding was of a similar magnitude to that elicited by insulin imprinting. Vanadate failed to increase the imprinting potential of insulin while ouabain even prevented insulin imprinting when administered together with insulin, but, did not affect imprinting when administered after insulin. By analogy with higher organisms it appears that inhibition of Na+K(+)-ATPase plays no role in the insulin-like effect of vanadate on the unicellular Tetrahymena, as judged also from the capacity to bind insulin of the generations of offspring.     

Effect of modification of membrane saccharides on hormonal imprinting in Tetrahymena

Concanavalin-A (Con-A) competes with insulin for the insulin binding sites of the receptor, an event resulting in overlapping bonds. If the saccharide components of the receptor are subjected to periodate treatment the binding of insulin decreases and so does the imprinting evoked by it. Simultaneously, the binding of Con-A increase immediately after the treatment and 24 hours following the insulin imprinting. This phenomenon indicates that though the two ligands (i.e. insulin and Con-A) overlap on the intact receptor, alterations in the saccharide component influence their binding though not in the same manner and not in the same direction. Periodate treatment, similarly as observed in mammalian lymphocyte cultures, enhanced the division of Tetrahymenas and the peak of Con-A binding occurred parallel to the peak of division after periodate treatment of equal duration. Periodate treatment disturbed not only the binding of Con-A but that of other lectins as well. It has been concluded that hormonal imprinting requires a physiological condition of the membrane and any disturbance of the membrane will lead to a decrease in the efficacy of imprinting.     

Ogataea thermophila sp. nov., the teleomorph of Candida thermophila

Ascospore formation was observed in the type strain of Candida thermophila Shin K-S, Shin YK, Yoon and Park on some yeast sporulation media. In addition, a further sporulating strain was found that proved to be conspecific with C. thermophila on the basis of sequences of the D1/D2 domain of the large-subunit (26S) rRNA gene and the internal transcribed spacer (ITS)1-5.8S rRNA gene - ITS2 region. Therefore, Ogataea thermophila Péter, Tornai-Lehoczki, Shin K-S & Dlauchy sp. nov. is proposed as the teleomorph of C. thermophila. The type strain is Y94(T)=JCM 10994(T)=KCCM 50661(T)=KCTC 17233(T).