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M Schleicher

Publications and source records attributed to M Schleicher.

At least 73 records · Page 4Linked to original sources
PubMed1991

Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum.

Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP. The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step. It has an apparent molecular mass of 44 kDa. Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0. Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM). These values are in good agreement with constants reported from other organisms. Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions. The enzyme is present throughout the developmental cycle of D. discoideum. Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library. The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase. Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron. The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli. Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.

Adenylosuccinate Synthase↗
PubMed1991

Phenotypes of cells with cytoskeletal mutations.

Analysis of the cytoskeleton has relied heavily on the identification of phenotypic alterations associated with mutations in cytoskeletal components. This approach has led to important findings for specific proteins. The last year has also strengthened the view that certain functions of the cytoskeleton are safeguarded by the presence of multiple protein forms.

Actin Cytoskeleton↗
PubMed1991

Domain structure in actin-binding proteins: expression and functional characterization of truncated severin.

Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.

Actins↗
PubMed1991

Dictyostelium discoideum contains two profilin isoforms that differ in structure and function.

Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a lambda gt11 cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13,064 Da vs 12,729 Da), has a more acidic isoelectric point (calc. pI 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most preparations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd 1.8 x 10(-6) M) as compared to that of profilin I (approx. Kd 5.1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Replacement of threonine residues by serine and alanine in a phosphorylatable heavy chain fragment of Dictyostelium myosin II.

The target sites of soluble myosin heavy chain kinases partially purified from growth phase or aggregation competent cells of Dictyostelium discoideum were identified by the use of normal and mutated fragments of the myosin heavy chain. The kinases from both developmental stages phosphorylated two previously established threonine residues, as well as an additional one. The newly identified site is located within the putative core region of the coiled-coil formed by the myosin tail. A lysine following the phosphorylated threonine residue is the only common feature of the sequences around these sites. The kinases, which specifically phosphorylate threonine residues in wild-type myosin, did accept serine if it was in the right structural context.

Alanine↗
PubMed1990

A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum.

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.

Amino Acid Sequence↗
PubMed1990

Severin is a gelsolin prototype.

A number of Ca2(+)-activated actin filament severing proteins have been identified in eukaryotic cells of diverse lineages. Gelsolin and villin, with molecular mass of about 80-90 kDa, and severin and fragmin, with molecular mass of about 40 kDa, have been isolated from vertebrates and invertebrates, respectively. We report here a direct comparison of the functional properties of gelsolin and severin, and the finding that the actin filament severing activity of severin, like that of gelsolin, is inhibited by polyphosphoinositides. However, severin does not nucleate actin filament assembly as well as gelsolin. These characteristics are very similar to those ascribed to the NH2-terminal half of gelsolin, supporting the idea that they are evolutionarily related. Regulation of severin by polyphospholipids raises the possibility that it may participate in agonist-stimulated regulation of the actin cytoskeleton in Dictyostelium discoideum.

Actins↗
PubMed1990

cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli.

A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from a lambda gt11 expression library and sequenced. The corresponding mRNA has a size of 0.7 kilobase and is down-regulated during early development of D. discoideum. Southern blotting demonstrated that the UMP-CMP kinase is encoded by a single gene. The deduced amino acid sequence of UMP-CMP kinase shows a high degree of homology with adenylate kinases from different sources with the highest degree of homology to cytosolic adenylate kinase from vertebrate muscle (43%). The enzyme expressed in Escherichia coli after cloning the cDNA into an ATG expression vector was purified and analyzed for its structural and kinetic properties. The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1 mM). The enzyme is strongly inhibited by the substrate analogue P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1 microM) and is inactivated by modification of free thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid).

Adenylate Kinase↗
PubMed1990

Heterodimeric capping proteins constitute a highly conserved group of actin-binding proteins.

The two subunits of the heterodimeric protein cap32/34, an actin-binding protein, are encoded by separate single-copy genes. We have established the genomic structure of both genes. A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences from Saccharomyces cerevisiae and Xenopus laevis show that heterodimeric capping proteins belong to a highly conserved group of actin-binding proteins. This conclusion is supported by the cross-reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes. In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34 promoter.

Actins↗
PubMed1990

Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases.

Developing cells of Dictyostelium discoideum contain crystalline inclusion bodies. The interlattice spaces of the crystals are approximately 11 nm, and their edge dimensions vary in aggregating cells from 0.1 to 0.5 micron. The crystals are enclosed by a membrane with the characteristics of RER. To unravel the nature of the crystals we isolated them under electron microscopical control and purified the two major proteins that cofractionate with the crystals, one of an apparent molecular mass of 69 kD, the other of 56 kD. This latter protein proved to be identical with the protein encoded by the developmentally regulated D2 gene of D. discoideum, as shown by its reactivity with antibodies raised against the bacterially expressed product of a D2 fusion gene. The D2 gene is known to be strictly regulated at the transcript level and to be controlled by cAMP signals. Accordingly, very little of the 56-kD protein was detected in growth phase cells, maximal expression was observed at the aggregation stage, and the expression was stimulated by cAMP pulses. The 69-kD protein is the major constituent of the crystals and is therefore called "crystal protein." This protein is developmentally regulated and accumulates in aggregating cells similar to the D2 protein, but is not, or is only slightly regulated by cAMP pulses. mAbs specific for either the crystal protein or the D2 protein, labeled the intracellular crystals as demonstrated by the use of immunoelectron microscopy. The complete cDNA-derived amino acid sequence of the crystal protein indicates a hydrophobic leader and shows a high degree of sequence similarity with Torpedo acetylcholinesterase and rat lysophospholipase. Because the D2 protein also shows sequence similarities with various esterases, the vesicles filled with crystals of these proteins are named esterosomes.

Amino Acid Sequence↗
PubMed1990

A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

Actins↗
PubMed1989

Alpha-actinin synthesis can be modulated by antisense probes and is autoregulated in non-muscle cells.

We used a 279 bp cDNA probe derived from a Dictyostelium alpha-actinin genomic sequence to assay the degree of homology between alpha-actinin from slime molds, mammalian and chicken cells. Recognition of this probe by vertebrate cells was shown in Southern and Northern blots, and by antisense RNA-induced depression of endogenous alpha-actinin synthesis in living cells. Micro-injection of Dictyostelium or chicken gizzard alpha-actinin resulted in incorporation of these proteins in stress fibers, peripheral microfilament belts and adhesion sites. Alpha-actinin-injected cells showed a marked, transient reduction of synthesis of the corresponding endogenous protein. These data emphasize the high degree of conservation of alpha-actinin during evolution and show for the first time autoregulation of synthesis for a microfilament protein.

Actin Cytoskeleton↗
PubMed1989

Ca2+-independent F-actin capping proteins. Cap 32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known actin-binding proteins.

Polyclonal and monoclonal antibodies against purified 32/34-kDa F-actin-capping protein from Dictyostelium discoideum were generated and used to isolate clones coding for both subunits from a lambda gt11 expression library. In addition genomic clones were isolated for the 34-kDa subunit. The sequences of the corresponding inserts were determined and the amino acid sequences of the proteins deduced. The amino acid sequences have been confirmed by sequencing tryptic peptides of both subunits. As judged from immunological data and sequence comparison, the subunits are completely different. Each of them is encoded by a single copy gene. The mRNAs are present throughout all stages of development. Severing proteins, which exert also capping activity, show no sequence similarity with the cap 32/34-kDa protein. However, a pronounced homology between the 32-kDa subunit and a hypothetical yeast protein was found indicating the presence of a protein homologous to the 32-kDa subunit of the D. discoideum capping protein in yeast.

Actin Depolymerizing Factors↗
PubMed1989

An extended ubiquitin of Dictyostelium is located in the small ribosomal subunit.

According to its cDNA sequence, the product of the DUB1 gene of Dictyostelium discoideum, called ubex52, consists of a ubiquitin monomer with a basic COOH-terminal tail of 52 amino acids that includes a putative zinc finger motif. Antipeptide antibodies raised against the COOH-terminal end of the tail indicated that the ubex52 protein is present in all developmental stages of D. discoideum and that similar proteins with apparent molecular masses of 15 to 17 kDa are found in yeast, wheat germ, Drosophila, and mammals. Subcellular fractionation showed that the D. discoideum and Saccharomyces cerevisiae proteins recognized by the antibodies are associated with the ribosomal fraction. After separation and purification of the 40 and 60 S ribosomal subunits of D. discoideum, the ubex52 protein was exclusively recovered in the small subunit.

Dictyostelium↗
PubMed1989

Hisactophilin, a histidine-rich actin-binding protein from Dictyostelium discoideum.

The purification, cloning, and complete cDNA-derived sequence of a 17-kDa protein of Dictyostelium discoideum are described. This protein binds to F-actin in a pH-dependent and saturable manner. It induces actin polymerization in the absence of Mg2+ or K+, and is enriched in the submembranous region of the amoeboid cells as indicated by immunofluorescence labeling of cryosections. The mRNA as well as the protein are present throughout growth and all stages of development. The protein is detected in both soluble and particulate fractions of the cells. From a plasma membrane-enriched fraction, minor amounts of the protein are stepwise solubilized with 1.5 M KCl, 0.1 M NaOH, and Triton X-100, but most of the protein is only solubilized with 1% sodium dodecyl sulfate. As judged by the apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gels, immunological cross-reactivity, and two-dimensional electrophoresis, the 17-kDa proteins from the soluble and particulate fraction resemble each other. The cDNA sequence does not reveal any signal peptide, trans-membrane region, or N-glycosylation site. Southern blots hybridized with a cDNA probe that spans the entire coding region show that the 17-kDa protein is encoded by a single gene. The most characteristic feature of the protein is its high content of 31 histidine residues out of 118 amino acids. We designate this protein as hisactophilin and suggest that this histidine-rich protein responds in its actin-binding activity to changes in cellular pH upon chemotactic signal reception.

Actins↗