GRASP65, a protein involved in the stacking of Golgi cisternae.

AbstractNEM prevents mitotic reassembly of Golgi cisternae into stacked structures. The major target of NEM is a 65 kDa protein conserved from yeast to mammals. Antibodies to this protein and a recombinant form of it block cisternal stacking in a cell-free system, justifying its designation as a Golgi ReAssembly Stacking Protein (GRASP65). One of the two minor targets of NEM is GM130, previously implicated in the docking of transport vesicles and mitotic fragmentation of the Golgi stack. GRASP65 is complexed with GM130 and is tightly bound to Golgi membranes, even under mitotic conditions when both are heavily phosphorylated. These results link vesicle docking, stacking of Golgi cisternae, and the disruption of both of these interactions during mitosis

Protein-chemical analysis of pertussis toxin reveals homology between the subunits S2 and S3, between S1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S2 as the haptoglobin-binding subunit

AbstractThe purified toxin of Bordetella pertussis was dissociated in 5 M urea in the presence of immobilized haptoglobin. The toxin was dissociated in free S1, free S5 and the free complexes S2-S4 and S3-S4, with S2-S4 as the only haptoglobin-binding moiety, identifying S2 as the haptoglobin-binding protein. Partial NH2-terminal amino acid sequences were obtained from the dissimilar toxin subunits, after separation by SDS-polyacrylamide gel electrophoresis followed by electroblotting onto polybrene-coated glass-fiber sheets. The sequences reveal extensive homology of the N-terminal portions of the constitutive subunits S2 and S3 and between S1 and the enterotoxin A chains of Vibrio cholerae and Escherichia coli

Amino-acid sequence analysis of proteins separated by one- and two-dimensional gel electrophoresis and electroblotted on polybase-coated glass-fiber sheets

Glass microfiber sheets to which guaternized ammonium polybases are adsorbed, were used as immobilizing membranes for protein electroblotting from sodium dodecylsulfate polyacrylamide gels. We have studied blotting efficiencies for various proteins, using different types of polybases and transfer buffers. We found that coating the glass-fiber sheets with poly(4-vinyl-N-methylpyridinium iodide) and carrying out the electrotransfer in Tris(hydroxymethyl)- aminomethane-boric acid buffer (50 mM each) provides the best conditions found so far. Until now, the NH2-terminal sequence of about one hundred different qlass-fiber immobilized proteins, polypeptides and glycoproteins, with molecular weight ranging from 10,000 to 150,000, have been successfully determined using classical gas -phase sequence analysis. The technique was found to be especially suitable for partial sequence analysis of proteins which were first enriched before final purification on qel..Moreover, we demonstrate that it is possible to determine the NH2~terminal amino acid sequence of the major proteins present in a total cellular extract, after separating them by 2D-PAGE followed by electroblotting on coated glass-fibeer. This allows one to analyse phenotypic alterations in cells or organisms at the elvel of the primary structure of the proteins. Based on the protein-sequence, a DNA-probe can be synthesized, which will allow the identification and isolation of the corresponding gene. This procedure was used to analyse the major proteins present in a total cellular extract of Nicotiana plumbaginifolia. Of the 10 proteins selected for analyses, eight yielded a sequence, ranging from 10 to 38 residues. Three of them are very homologous with other eukaryotic proteins: triosephosphate isomerase, Mn-superoxide dismutase and 1,3-beta-glucanase

Protein-blotting on Polybrene-coated glass-fiber sheets: a basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel

A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350–4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The later procedure was found to be more efficient and yielded binding capacities of ± 20 μ/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (± 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel

Auxin binding proteins from maize coleoptiles : purification and molecular characterization

To understand precisely the mechanisms by which hormones like auxins regulate plant differentiation and development, it is essential to isolate putative hormone receptors. We have purified the major auxin binding protein from maize coleoptiles to homogeneity. The protein has an apparent molecular weight of 22,000 Da and binds 1-naphthylacetic acid with a KD of 2.4 x 10(-7) M. Protein sequence analysis allowed the construction of oligonucleotide probes to isolate a corresponding cDNA coding for this protein. The open reading frame of this cDNA predicts a protein of 201 amino acids and 21,990 Da in size. The amino acid sequence includes a cleavable N-terminal signal sequence and a C-terminal signal element consisting of the amino acids Lys Asp Glu Leu known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum