Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Mass selection of conditional mating mutants of Tetrahymena thermophila

The mating reaction in Tetrahymena thermophila includes a starvation period and two distinct cell interactions, co-stimulation and cell pairing, before the cells are cytoplasmically joined as conjugants. A selection procedure for harvesting mutants unable to mate at a restrictive temperature has been developed. A conjugant pair consisting of one cycloheximide-resistant cell and one wild-type cell (cycloheximide-sensitive) was itself sensitive to the drug. By adding cycloheximide and nutrient medium to a cross made at the restrictive and grow. Repetition of the selection procedure enriched for cells unable to conjugate at the restrictive temperature. The selected cells were able to grow at 38 degrees C and could conjugate at 28 degrees C. This procedure may be narrowed to select specifically for cell interaction mutants.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: starved versus nonstarved.

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent ("initiated") cells were compared with those from non-starved, mating-incompetent ("non-initiated") cells, by gel electrophoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glycoconjugate as having a potential role in control of pair formation during mating.     

Role for endocytosis in conjugation in Tetrahymena

We examined the effect of inhibitors of receptor-mediated endocytosis on cell pair formation during conjugation in Tetrahymena thermophila. Dansylcadaverine (20 microM), methylamine (20 mM), and bacitracin (2 mg/ml) prevented cell pair formation even when added poststarvation, after mixing of cells of opposite mating types (during the preparing interaction). Chloroquine (10 and 25 microM) did not inhibit cell pair formation, leading to the conclusion that inhibition by dansylcadaverine, methylamine, and bacitracin is not due to an alkalinization of the lysosome. These results did not allow us to define the time in the preparing interaction at which inhibition occurs, nor to identify the cellular components involved, but they did support the hypothesis that an endocytotic event(s) plays a role in the cell contact-mediated recognition which occurs during the preparing interaction.     

Locus-dependent profiles of the rescue of nonexcitable behavioral mutants during conjugation in Tetrahymena thermophila.

The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about 1 h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2 h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB, more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca2+ channels.     

Differential Expression and Functionality of TRPA1 Protein Genetic Variants in Conditions of Thermal Stimulation

The role of genetic modifications of the TRPA1 receptor has been well documented in inflammatory and neuropathic pain. We recently reported that the E179K variant of TRPA1 appears to be crucial for the generation of paradoxical heat sensation in pain patients. Here, we describe the consequences of the single amino acid exchange at position 179 in the ankyrin repeat 4 of human TRPA1. TRPA1 wild type Lys-179 protein expressed in HEK cells exhibited intact biochemical properties, inclusive trafficking into the plasma membrane, formation of large protein complexes, and the ability to be activated by cold. Additionally, a strong increase of Lys-179 protein expression was observed in cold (4 °C) and heat (49 °C)-treated cells. In contrast, HEK cells expressing the variant Lys-179 TRPA1 failed to get activated by cold possibly due to the loss of ability to interact with other proteins or other TRPA1 monomers during oligomerization. In conclusion, the detailed understanding of TRPA1 genetic variants might provide a fruitful strategy for future development of pain treatments.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Conjugal blocks in Tetrahymena pattern mutants and their cytoplasmic rescue. I. Broadened cortical domains (bcd).

The cortical pattern mutant broadened cortical domains (bcd) in Tetrahymena thermophila is unable to complete the nuclear events associated with conjugation. bcd x bcd pairs become arrested at the "nuclear exchange" configuration. Genetic analysis reveals that the bcd conjugal block is 100% penetrant, under macronuclear control, and rescueable (a) by outcrossing to a wild-type partner, (b) by administration of a hyperosmotic shock 5 hr after cells are mixed for mating, or (c) by cytoplasmic transfusion from a wild-type donor. Cytological analysis reveals that the conjugal block is primarily the result of failure in pronuclear fusion (karyogamy). bcd pairs also exhibit reduced nuclear exchange efficiency and a failure of macronuclear anlagen formation. The hypothesis is proposed that the bcd+ gene codes for a microtubule-based organelle "motor" similar to kinesin.     

The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila

A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.     

Oral assembly in left-handed Tetrahymena thermophila

We have investigated oral development in a non-genetically derived left-handed (LH) form of Tetrahymena thermophila, in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells' anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180 degrees rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama. We present a model for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

The role of Glu39 in MnII binding and oxidation by manganese peroxidase from Phanerochaete chrysoporium

Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of the extracellular lignin degrading system in these organisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn(II) and efficiently release Mn(III). Initial site-directed mutagenesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-type Mn binding and rate of oxidation, calling into question the role of E39 as an Mn ligand. To investigate this hypothesis, we performed computer modeling studies which indicated metal-ligand bond distances in the E39D variant and in an E35D--E39D--D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D variants used in the previous study, as well as an E39A single variant and the E35D--E39D--D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn(II) binding (K(m) increases 20--30-fold) and Mn(II) oxidation (k(cat) decreases 50--400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The catalytic efficiency of the E39A variants decreased approximately 10(4)-fold, while that of the E39D variant decreased approximately 10(3)-fold. Contrary to initial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of the triple variant decreased only 10(4)-fold, which is approximately 10(2)-fold better than that reported for the E35Q--D179N double variant. These combined studies indicate that precise geometry of the Mn ligands within the Mn binding site of MnP is essential for the efficient binding, oxidation, and release of Mn by this enzyme. The results clearly indicate that E39 is a Mn ligand and that mutation of this ligand decreases both Mn binding and the rate of Mn oxidation.     

Oral Assembly in Left-Handed Tetrahymena thermophila

We have investigated oral development in a non-genically derived left-handed (LH) form of Tetrahymena thermophila , in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells'anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a "sculptured" appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180° rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama [36, 37]. We present a model (Fig. 37) for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for "secondary" OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.     

Karyonidal inheritance of mating types in tetrahymena malaccensis

Mating type determination in Tetrahymena malaccensis is karyonidal, ie, the four new macronuclei developing in a single conjugating pair are independently determined as to which of the six known mating types they will express. Occasional selfing clones are similar to those in T thermophila, in that any one is capable of stabilizing at a restricted range of mating types. The genetic basis of mating type potentialities is incompletely resolved. T malaccensis may, like T thermophila and T canadensis, have a single multiallelic locus that controls the array of types. Quantitative considerations suggest, however, that other loci may be involved.     

Phosphorylation of a threonine unique to the short C-terminal isoform of betaII-spectrin links regulation of alpha-beta spectrin interaction to neuritogenesis

Spectrin tetramers are cytoskeletal proteins required in the formation of complex animal tissues. Mammalian alphaII- and betaII-spectrin subunits form dimers that associate head to head with high affinity to form tetramers, but it is not known if this interaction is regulated. We show here that the short C-terminal splice variant of betaII-spectrin (betaIISigma2) is a substrate for phosphorylation. In vitro, protein kinase CK2 phosphorylates Ser-2110 and Thr-2159; protein kinase A phosphorylates Thr-2159. Antiphospho-Thr-2159 peptide antibody detected phosphorylated betaIISigma2 in Cos-1 cells. Immunoreactivity was increased in Cos-1 cells by treatment with forskolin, indicating that phosphorylation is promoted by elevated cAMP. The effect of forskolin was counteracted by the cAMP-dependent kinase inhibitor, H89. In vitro, protein kinase A phosphorylation of an active fragment of betaIISigma2 greatly reduced its interaction with alphaII-spectrin at the tetramerization site. Mutation of Thr-2159 to alanine eliminated inhibition by phosphorylation. Among the processes that require spectrin in mammals is the formation of neurites (incipient nerve axons). We tested the relationship of spectrin phosphorylation to neuritogenesis by transfecting the neuronal cell line, PC12, with enhanced green fluorescent protein-coupled fragments of betaIISigma2-spectrin predicted to act as inhibitors of spectrin tetramer formation. Both wild-type and T2159E mutant fragments allowed normal neuritogenesis in PC12 cells in response to nerve growth factor. The mutant T2159A inhibited neuritogenesis. Because the T2159A mutant represents a high affinity inhibitor of tetramer formation, we conclude that tetramers are requisite for neuritogenesis. Furthermore, because both the T2159E mutant and the wild-type allow neuritogenesis, we conclude that the short C-terminal betaII-spectrin is phosphorylated during this process.     

Characterization of a Tetrahymena thermophila mutant strain unable to develop normal thermotolerance.

For Tetrahymena thermophila cells to survive extended periods of time at 43 degrees C, they must continuously synthesize heat shock proteins. For its translational machinery to function at 43 degrees C, T. thermophila requires either prior nonlethal heat shock treatment or brief treatment with partially inhibiting doses of cycloheximide or emetine. We have identified and characterized a mutant strain of T. thermophila (MC-3) in which prior nonlethal heat shock does not prevent protein synthesis inactivation at 43 degrees C. In addition, treatment of MC-3 cells with either of the antibiotics that normally confer 43 degrees C thermoprotection on wild-type cells elicited no similar thermoprotective response in these cells. Despite these phenotypic characteristics, by other criteria MC-3 synthesized a normal, functional array of heat shock proteins at 40 degrees C, a nonlethal heat shock protein-inducing temperature. The mutation in MC-3 which prevents the thermostabilization of protein synthesis by nonlethal heat shock is, by genetic criteria, most likely the same one which prevents the induction of thermotolerance by drug treatments. We present evidence that this mutation may affect some ribosome-associated functions.     

Enhanced proliferative effects of a baculovirus-produced fusion protein of insulin-like growth factor and alpha(1)-proteinase inhibitor and improved anti-elastase activity of the inhibitor with glutamate at position 351

Alpha(1)-proteinase inhibitor (API) was coupled at the C-terminus of a human insulin-like growth factor (IGF) analog to facilitate its production in insect cells. This fusion protein significantly increased thymidine incorporation into HL-60 cells as compared with the incorporation observed with an equivalent molar mixture of the IGF analog and API. The M351E variant of API has been previously shown to reduce aggregate formation in prokaryotic expression systems. When the oxidation-sensitive methionine 351 of the inhibitor was changed to glutamate, the M351E variant was secreted in larger amounts from insect cells than the corresponding fusion protein with wild-type API. The M351E fusion protein and the corresponding chimera containing the wild-type API were tested for their capacity to inhibit human neutrophil elastase. The M351E variant was a more potent elastase inhibitor than the fusion protein containing the wild-type analog, whereas the proliferative activity of both chimeras was identical. The described mitogenic effect of the chimera and the improved anti-elastase activity of the M351E variant are two ideal properties for therapeutic agents acting in pathological situations where cell proliferation and inhibition of neutrophil elastase have to take place simultaneously, such as during wound healing.     

Impaired tetramer assembly of variant medium-chain acyl-coenzyme A dehydrogenase with a glutamate or aspartate substitution for lysine 304 causing instability of the protein.

Ninety percent of variant medium-chain acyl-CoA dehydrogenase (MCAD) alleles in patients with MCAD deficiency carry a 985 A-->G transition which causes glutamate substitution for lysine 329 in precursor (p) MCAD (K-304 in mature MCAD). We have used site-directed mutagenesis to produce three variant cDNAs encoding variant pMCAD with glutamate (Kp329E2), aspartate (Kp329D), or arginine (Kp329R) substitution for Kp329. We carried out in vitro expression of cDNAs, and incubated the translation products with isolated rat liver mitochondria. Kp329E was imported into mitochondria and processed into the mature subunit as efficiently as wild-type. Gel filtration analysis of the mitochondria revealed that at 10 min after import, markedly more K304E eluted as a monomer than did wild-type, and the amount of K304E tetramer formed was distinctly less than wild-type at any point up to 60 min after import, indicating that the assembly of K304E is defective. After further incubation, K304E decayed more rapidly than did wild-type, indicating a reduced stability. In similar studies, K304R behaved like the wild-type, while K304D closely resembled K304E, indicating that the presence of a basic residue at 304 is essential for tetramer formation and intramitochondrial stability of mature MCAD.