Anton Schweiger

Stress-induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine-53 adjacent to the DNase I binding loop

Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyostelium discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger Mono‐P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress.

Classification of tyrosine kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains

Two new kinases of Dictyostelium discoideum were identified by screening of a λgt11 expression library with a phosphotyrosine specific antibody. Amino‐acid sequences derived from cDNA and genomic clones indicate that DPYK3 is a protein of 150 kDa and DPYK4, a protein of 75 kDa. The C‐terminal fragments of each protein were produced in Escherichia coli and shown to be autocatalytically phosphorylated at tyrosine residues. A common feature of these kinases is the presence of two different sequence stretches in tandem that are related to kinase catalytic domains. The sequence relationships of DPYK3 and 4 to other protein kinases, and the positions of their catalytic domain sequences within the phylogenetic tree of protein kinases were analysed. Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two previously described tyrosine kinases of D. discoideum, a branch of their own, separate from the tyrosine kinase domains in sensu strictu. Domain II in DPYK4 is found on a different branch close to serine/threonine kinases.

Proteins associated with rapidly-labelled nuclear RNA and their occurrence in rat-liver cytoplasmic fractions☆

Abstract 1. Free-flow electrophoresis was used for the group separation of proteins from crude nuclear saline extracts. In the presence of 4.0 M urea, a purified preparation of slightly acidic informosome protein was obtained after two successive electrophoretic runs. 2. Using the same procedure, significant amounts of informosome protein were also isolated from subfractions of free polyribosomes and rough microsomes of rat liver. In addition to its typical distribution during free-flow electrophoresis, the protein was identified by polyacrylamide disc electrophoresis at pH 4.5. The results may indicate that nuclear informosome protein is transferred with RNA to cytoplasmic sites with target function for mRNA.

Identical properties of an mRNA-bound protein and a cytosol protein with high affinity for polyadenylate.

Abstract A rat liver soluble protein which binds very tightly to polyadenylate in vitro, and a specific mRNA-bound protein from free polyribosomes, have been isolated by techniques of affinity chromatography. The two protein preparations were compared on the basis of criteria such as stability and specificity of the binding, molecular weight, which was 78,000 in both cases, and amino acid composition. We conclude that the two proteins are identical or at least highly related species and that the cytosol component may be a precursor of the polyribosome-associated protein.

Isolation of RNA-binding proteins from rat liver nuclear 30 S-particles

In order to detect the significance of the relatively large proportion of protein of nuclear 30 S-particles which accounts for about 80% of the particle weight [l] several investigations have been performed [2-S]. Recently, it has been shown that the protein moiety comprises enzyme components involved in the pr@ cessing of DNA-like RNA [6]. Another inherent property of 30 Sparticles is their ability to bind extra RNA added in vitro [7]. This reaction possibly reflects a function of the particles in vivo and studies on it further offer an approach to certain aspects of their structural organisation. The binding may be explained from two principal modes of interaction: (i) The affmity to DNA-like RNA depends on the complex quaternary structure [g] of the whole intact particle or (ii) is conferred to the particle by some of its subunits or components, most likely proteins [9]. The results presented in this report are in favour of the second alternative. They show that the RNA-binding property of the particles can be attributed to extractable proteins which are highly active in the RNA-binding assay described previously [ lo] . The isolated proteins had molecular weights of 25 000 to 42 000. From studies on the in vitro incubation of the particles in the presence of [32P] ATP it appeared that the extractable material also contained one or more phosphoprotein components.

Phosphotyrosine-containing proteins in Dictyostelium discoideum

Phosphotyrosine‐containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and inununoprecipitation using a monoclonal anti‐phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205–220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [γ‐32p]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205–220 kDa bands.

Studies on the RNA-binding activity of rat liver cytosol

Abstract Labelled RNAs from rat liver nuclei or yeast were incubated with protein fractions derived from rat liver cytoplasm and the amount of protein-bound RNA was measured in a filter assay using cellulose nitrate membranes. 1. 1. An “RNA-binding factor” which is rather labile and tends to form insoluble aggregates was purified about 80-fold with the aid of free-flow electrophoresis and density gradient centrifugation at a pH of 8.5. 2. 2. The binding factor is confined to the high-speed supernatant of the cytoplasm and the purified preparation has a sedimentation coefficient of 9 S. It is free from nucleic acids and shows slightly basic properties. 3. 3. From its behaviour during the purification, it is concluded that the binding factor consists of not more than a few components which are possibly arranged in a complex structure. Disc electrophoresis at pH 4.5 of the most purified preparation revealed three discrete bands showing similar electrophoretic mobilities as Bands A, B, and C of the protein of nuclear informosomes. 4. 4. The binding reaction is presumably a nonenzymatic process mediated by electrostatic forces as it proceeds at a high velocity independent of temperature between 0 and 37°. 5. 5. In the presence of inorganic phosphate (0.01 M), SH-blocking chemicals as p-chloromercuribenzoate (10−4 M), sodium deoxycholate or Triton WR 1339 (1%), the formation of the complex was inhibited.

Mammalian proteins with affinity to polynucleotides: Isolation by affinity chromatography from rat liver cytosol and nucleosol

The mechanism of nucleo-cytoplasmic transport of mRNA in eukaryotic cells is still little understood especially in details concerning the participation of proteins and their significance or function in this process. It has been suggested that nuclear 30 Sparticles or components there of are involved as vehicle protein [l-4] or that mRNA and its cyto: plasmic precursor are associated with proteins which are not derived from nuclear particles and appear to be changed at certain stages of the life cycle of the RNA [3,5]; In the case of mRNA-protein structures of mammalian polyribosomes a protein with a mol. wt. of 78 000 has been shown to be bound to the polyadenylate segment of the RNA [6] which fact indicates that this region may serve as a binding site for specific proteins. The idea that various of the proteins mentioned above may not exist exclusively as ribonucleoproteins but also in the RNA-free state has been promoted by the finding that the soluble fraction of nuclear or cytoplasmic extracts of eukaryotic cells contain ‘RNA-binding’ proteins [7-lo]. Most attempts, however, to reveal such similarities or even identity were complicated by difficulties in the isolation of the binding proteins. In the present report a procedure is described which allows the separation of soluble binding proteins from rat liver nuclei and cytoplasm on the basis of their affinity to Sepharose 4B-polyadenylate. Two fractions were obtained at KC1 concentrations of 0.05 and 1 .O M. In most experiments poly(A) was used in the affinity chromatography because of seeral advantages

Identification of a 110 000 molecular weight protein associated with heterogeneous nuclear RNA and messenger RNA in rat liver cells

Abstract The composition of proteins associated with heterogeneous nuclear RNA (hnRNA) and polyribosomal messenger RNA (mRNA) in rat liver cells has been studied using sodium dodecylsulfate (SDS)-plate gel electrophoresis. The nuclear RNP was isolated as the 30–40 S monomer by means of several different procedures including extraction of nuclei at 0 and 25 °C and ultrasonic treatment. These preparations were shown to contain the same set of specific proteins when analysed electrophoretically. Dissociation of free polyribosomes was accomplished in the presence of either EDTA or puromycin at high ionic strength and the mRNP separated on columns of oligo(dT) cellulose. Two to three proteins with identical molecular weights were identified in the SDS band patterns of both hnRNP and mRNP; in particular a 110000 D double-band was most conspicuous in both band patterns.

Zur identifizierung von polysacchariden nachwies der zuckerbausteine durch dünnschicht-chromatographie und infrarotspektroskopie

Abstract A method for the identification of sugars and uronic acids as components of polysaccharides of various origin was developed. The sample (e.g. pure substances, mucopolysaccharides of muscle or additives to food) was hydrolyzed by a cation exchange resin. The resulting mixture of compounds was separated on a cellulose thin layer plates. Isolated bands of sugars were pressed into KBr disks after elution and identified by their infrared spectra. The separated uronic acids were converted into their cinchonic salts, whose infrared spectra allowed a differentiation of d -glucuronic, d -galacturonic and d -mannuronic acid.