Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

Abstract

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin‐myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown‐shaped cell surface projections of growth‐phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino‐terminal domain with similarities to the beta subunits of G proteins and a carboxy‐terminal domain with a high tendency for alpha‐helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

“…When CF-was substituted by glutamate to approximate physiological conditions (de Hostos et al, 1991), no inhibition of the binding up to 100 mM potassium glutamate was observed; at 75 mM potassium glutamate the binding was even enhanced. An equilibrium binding constant of Dd plastin with F-actin was determined by high-speed centrifugation in the presence of EGTA ( Figure 3C).…”

“…Purification of a 67-kDa Protein (p67) from a Contracted Actin-Myosin Complex Contracted actin-myosin complex was prepared from cells starved for 12 h essentially as described previously (Condeelis and Taylor, 1977;Fechheimer and Taylor, 1984;de Hostos et al, 1991). Washed cells were resuspended at 4°C in an equal volume of homogenization buffer [5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 5 mM ethylene glycol-bis(,B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (PIC) consisting of leupeptin, bestatin, pepstatin A, antipain, 10 ,ug/ml each] and disrupted by nitrogen decompression in a Parr bomb (800 psi), followed by gentle agitation with a loose fitting Dounce homogenizer.…”

“…After 1 h on ice, the contracted actin-myosin gel was washed twice with homogenization buffer (pH 7.5) supplemented with MgCl2, KCl, and ATP, resuspended, and dialyzed against HEPES-G buffer [5 mM HEPES, pH 7.5, 0.2 mM dithiothreitol (DTT), 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM MgCl2, PIC (1 gg/ml of each inhibitor), 1 mM benzamidine]. Upon fractionation of the dissolved proteins on a DE52 column, p67 eluted in a linear gradient at 75-100 mM NaCl, overlapping with coronin (de Hostos et al, 1991) and a 54-kDa polypeptide, which was most likely a degradation product. Its amount could be reduced by minimizing proteolysis.…”

DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum

“…Southern and Northern blots were hybridized in the presence of 50% formamide, and Western blots performed after SDS-PAGE of total cellular proteins in 10% gels. The Western blots were labeled using hybridoma 216-394-1 or 176-2-5 [8] culture supernatant and iodinated sheep antimouse IgG (Amersham, Braunschweig, Germany). For quantification of DGAP1 overexpression, antibody 12~I-label on the 95 kDa band of DGAP1 was determined with a Fuji Phosphoimager.…”

Phosphorylation of Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-specific Kinase, TSSK-2.

“…WD repeats typically contain a GH dipeptide 11-24 residues from the N-terminus, and the WD dipeptide at the C-terminus. Proteins with WD repeats have diverse functions, such as signal transduction, RNA processing, transcription, cytoskeleton assembly and mitotic-spindle formation, regulation of vesicle formation and vesicular trafficking, control of various aspects of cell division, and the regulation of sulfur metabolism in fungi [9][10][11][12][13][14][15][16][17][18].…”