Interaction of sulfhydryl reactive reagents with components involved in (1,3)-β-glucan synthesis from Candida albicans

1995 ◽  
Vol 41 (8) ◽  
pp. 692-698 ◽  
Author(s):  
David J. Frost ◽  
Kim Brandt ◽  
Tim Kaufmann ◽  
Robert Goldman
Keyword(s):  
Candida Albicans ◽  
Binding Protein ◽  
Sulfhydryl Reagents ◽  
Gtp Binding Protein ◽  
Rapid Separation ◽  
Glucan Synthase ◽  
Gtp Binding ◽  
Catalytic Active Site ◽  
Membrane Bound ◽  
Glucan Synthesis

Glucan synthesis was sensitive to several sulfhydryl reacting compounds: mercurials, reversible disulfides, and an alkylating sulfhydryl reagent (IC50 3–45 μM). Thiol groups associated with glucan synthesis were hydrophilic in nature, since both hydrophilic and hydrophobic reagents were active. Glucan synthase complex consists of at least two components: a peripheral GTP-binding protein that can be solubilized with detergents (supernatant) and the catalytic membrane-bound component (pellet). A rapid separation technique was developed to study sulfhydryl interactions with the complex. The GTP-binding protein was solubilized with 0.6% 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate from isolated microsomes of Candida albicans cells grown at either 10 or 30 °C. The residual membranous fraction contained the core catalytic moiety of glucan synthase. Both fractions were devoid of glucan synthase activity until they were reconstituted by mixing the two fractions together. In reconstitution experiments, the pellet lost almost 50% activity when preincubated with 2.5 μM N-ethylmaleimide and combined with an untreated supernatant whereas only 10% activity was lost when the supernatant was treated with N-ethylmaleimide. The catalytic active site of glucan synthase was not protected with UDP-Glc when preincubated with 10 μM N-ethylmaleimide but the GTP-binding fraction was partially protected with GTPγS.Key words: Candida albicans, (1,3)-β-glucan synthase, GTP-binding proteins, solubilization, sulfhydryl reagents.

scholarly journals The GTP-binding Protein Rho1p Is Required for Cell Cycle Progression and Polarization of the Yeast Cell

1999 ◽  
Vol 146 (2) ◽  
pp. 373-387 ◽  
Author(s):  
Jana Drgonová ◽  
Tomás Drgon ◽  
Dong-Hyun Roh ◽  
Enrico Cabib
Keyword(s):  
Cell Cycle ◽  
Yeast Cell ◽  
Cell Cycle Progression ◽  
Binding Protein ◽  
Cell Polarization ◽  
Nonpermissive Temperature ◽  
Gtp Binding Protein ◽  
Cycle Progression ◽  
Glucan Synthase ◽  
Gtp Binding

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of β(1→3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1E45I in the G1 stage of the cell cycle did not bud at 37°C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1V43T and rho1F44Y, showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1E45I when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1E45I. Nuclear division did not occur in the mutant at 37°C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of β(1→3)glucan synthase in rho1 E45I, although diminished at 37°C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and β(1→3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.

THE ROLE OF INOSITIDES, PHOSPHOLIPASE C AND G-PROTEINS IN RECEPTOR TRANSDUCTION

1987 ◽  
Author(s):  
Eduardo G Lapetina
Keyword(s):  
Phospholipase C ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Cytosolic Phospholipase ◽  
Gtp Binding ◽  
Membrane Bound ◽  
Stimulation Of

It is now widely recognized that the activation of phospholipase C by specific agonists leads to the formation of two second messengers: (1) inositol trisphosphate, which releases Ca2+ from the endoplasmic reticulum to the cytosol and (2) 1,2- diacylglycerol, which stimulates protein kinase C. In the past few years, GTP-binding proteins have been associated with the regulation of phospholipase C. However, the identity of the GTP-binding protein involved and the type of association with phospholipase C is not yet known. It is now recognized that there are two types of phospholipase C enzymes: (a) a soluble enzyme that has been characterized in several tissues and does not preferentially hydrolyze polyphospholinositides and (b) membrane-bound enzymes that are coupled to the receptors, specifically hydrolyzing polyphosphoinositides and activated by membrane guanine nucleotide-binding proteins. Recent reports have tried to assess the involvement of GTP-binding proteins in the agonist-induced stimulation of phospholipase C, and various related aspects have been reported. These are concerned with: (a) detection of various GTP-binding proteins in platelets, (b) the effects of known inhibitors of GTP-binding proteins such as GDPgS or pertussis toxin on the agonist-induced stimulation of phospholipase C, (c) the direct effects of stimulators of GTP-binding proteins such as GTP, GTP-analogs and fluoride on phospholipase C activity, (d) the possible association of GTP-binding proteins to cytosolic phospholipase C that would then lead to degradation of the membrane-bound inositides and (e) cytosolic phospholipase C response to the activation of cell surface receptors. The emerging information has had contradictory conclusions. (1) Pretreatment of saponin-permeabilized platelets with pertussis toxin has been shown to enhance and to inhibit the thrombin-induced activation of phospholipase C. Therefore, it is not clear if a G protein that is affected by pertussis toxin in a manner similar to Gi or Go plays a central role in activation of phospholipase C. (2) Studies on the effect of GDPβ;S are also conflicting indicating that there may be GTP-independent and/or -dependent pathways for the activation of phosphoinositide hydrolysis. (3) A cytosolic phospholipase C is activated by GTP, and it has been advanced that this activity might trigger the hydrolysis of membrane-bound inositides. A cytosolic GTP-binding protein might be involved in this action, and it is speculated that an α-subunit might be released to the cytoplasm by a receptor-coupled mechanism to activate phospholipase C. However, no direct evidence exists to support this conclusion. Moreover, the exact contribution of phospholipase C from the membranes or the cytosol to inositide hydrolysis in response to cellular agonists and the relationship of those activites to membrane-bound or soluble GTP-binding proteins are unknown. Our results indicate that the stimulation of phospholipase C in platelets by GDPβS and thrombin are affected differently by GDPβS. GDPgSinhibits the formation of inositol phosphates produced by GTPγS but not that induced by thrombin. Thrombin, therefore, can directly stimulate phospholipase C without the involvement of a “stimulatory” GTP-binding protein, such as Gs, for the agonist stimulation of adenylate cyclase. However, an “inhibitory” GTP-binding protein might have some influence on thrombin-stimulated phospholipase C, since in the presence of GDPγS thrombin produces a more profound stimulation of phospholipase C.This “inhibitory” GTP-binding protein might be ADP-ribosylated by pertussis toxin because pertussis toxin can also enhance thrombin action on phospholipase C activity. Therefore, phospholipase C that responds to thrombin could be different from the one that responds to GTPγS. Cytosolic phospholipase C can be activated by GTP or GTP analogs, and the one that responds to thrombin should be coupled to the receptors present in the plasma membrane. The initial action of thrombin is to directly activate the plasma membrane-bound phospholipase C and the mechanism of this activation is probably related to the proteolytic action of thrombin or the activation of platelet proteases by thrombin. In agreement with this, trypsin can also directly activate platelet phospholipase C and, subsequently, GTPyS produces further activation of phospholipase C. If these two mechanisms are operative in platelets, the inhibition of cytosolic phospholipase C by GDPβS would allow a larger fraction of inositides for degradation of the thrombin-stimulated phospholipase C, as our results show.

NOVEL GTP-BINDING PROTEINS OF CYTOSOLIC AND MEMBRANE FRACTIONS OF HUMAN PLATELETS

1987 ◽  
Author(s):  
Eduardo G Lapetina ◽  
Bryan R Reep ◽  
Luis Molina Y Vedia
Keyword(s):  
Phospholipase C ◽  
Binding Proteins ◽  
Binding Protein ◽  
Human Platelets ◽  
Trypsin Treatment ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Sds Page ◽  
Membrane Bound

We have assessed the binding of (α-32P)GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with (α-32p)GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP or GTPyS and by 100 nM GDP; binding was unaffected by 1 nM-1 μM ATP. One main GTP-binding protein (29.5 KDa) was detected in the membrane fraction while three others (29, 27, and 21 KDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 KDa) were degraded by trypsin; another cytosolic protein (21 KDa) and the membrane-bound protein (29.5 KDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of (α-32P)GTP to the membrane-bound protein. GTPyS still stimulates phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTPyS. We have started to purify the membrane-bound, trypsin-resistant, GTP-binding protein. Purification includes 1 M NaCl extraction and the use of an FPLC system with successive phenyl superose and superose 12 columns.

Light-modulated subcellular localization of the alpha-subunit of GTP-binding protein Gq in crayfish photoreceptors

1996 ◽  
Vol 13 (3) ◽  
pp. 539-547 ◽  
Author(s):  
Akihisa Terakita ◽  
Hideki Takahama ◽  
Satoshi Tamotsu ◽  
Tatsuo Suzuki ◽  
Takahiko Hariyama ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Binding Protein ◽  
Light Condition ◽  
Alpha Subunit ◽  
Soluble Form ◽  
Acid Modification ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Membrane Bound

AbstractGq-type GTP-binding protein (Gq) plays an important role in invertebrate visual phototransduction. The subcellular localization of the alpha subunit of visual Gq in crayfish photoreceptor was investigated immunocytochemically and biochemically to demonstrate the details of the rhodopsin-Gq interaction. The localization of Gq(alpha) changed depending on the light condition. In the dark, Gq(alpha) was localized in the whole rhabdoms as the membrane-bound form. In the light, half of the Gq(alpha) was localized in the cytoplasm as the soluble form. The translocation of Gq(alpha) was reversible. The light-modulated translocation possibly controls the amount of Gq that can be activated by rhodopsin. In vitro hydroxylamine treatment of rhabdomeric membranes suggested that the translocation was regulated by the fatty-acid modification of Gq(alpha).

scholarly journals Cellular Accumulation, Localization, and Activity of a Synthetic Cyclopeptamine in Fungi

1998 ◽  
Vol 42 (2) ◽  
pp. 389-393 ◽  
Author(s):  
John O. Capobianco ◽  
Dorothy Zakula ◽  
David J. Frost ◽  
Robert C. Goldman ◽  
Leping Li ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Glucan Synthase ◽  
Cellular Accumulation ◽  
Drug Concentrations ◽  
Drug Reservoir ◽  
Membrane Bound ◽  
Glucan Synthesis ◽  
Major Drug

ABSTRACT A novel synthetic cyclopeptamine, A172013, rapidly accumulated by passive diffusion into Candida albicans CCH442. Drug influx could not be totally facilitated by the membrane-bound target, β-(1,3)-glucan synthase, since accumulation was unsaturable at drug concentrations up to 10 μg/ml (about 1.6 × 10−7molecules/cell), or 25× MIC. About 55 and 23% of the cell-incorporated drug was associated with the cell wall and protoplasts, respectively. Isolated microsomes contained 95% of the protoplast-associated drug, which was fully active against glucan synthesis in vitro. Drug (0.1 μg/ml) accumulation was rapid and complete after 5 min in several fungi tested, including a lipopeptide/cyclopeptamine-resistant strain of C. albicans(LP3-1). The compound penetrated to comparable levels in both yeast and hyphal forms of C. albicans, and accumulation inAspergillus niger was 20% that in C. albicans. These data indicated that drug-cell interactions were driven by the amphiphilic nature of the compound and that the cell wall served as a major drug reservoir.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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