Substrate specificity of peptide permeases in Candida albicans

Abstract Specificity of peptide transport systems in Candida albicans was studied using as an experimental tool novel anticandidal peptides, containing the N³-4-methoxyfumaroyl- l -2,3-diamino-propanoic acid residue. Studies on cross-resistance and on peptide uptake by spontaneous mutants resistant to toxic peptides, confirmed the multiplicity of peptide permeases in Candida albicans . At least two peptide permeases exist in this microorganism; the first one, specific for di- and tripeptides and the second, for oligopeptides containing 3–6 amino acids. The rate of the tritetra tetra-, penta- and hexapeptide transport in the mycelial form of Candida albicans is about 2-times higher than in the yeast form, while that of dipeptides is markedly reduced.
Tripeptides are proposed as the most efficient carriers for the delivery of 'warhead' amino acids into Candida albicans cells.     

Kinetics and concentration dependency of cAMP-induced desensitization of a subpopulation of surface cAMP receptors in Dictyostelium discoideum

Extracellular cAMP induces the rapid activation of guanylate cyclase, which adapts within 10 s to constant cAMP concentrations. A new response can be induced either by a higher cAMP concentration or by the same cAMP concentration at some time (t1/2 = 90 s) after removal of the previous stimulus. Stimulation of guanylate cyclase is supposed to be mediated by a subpopulation of cell surface cAMP receptors (B-sites). These sites can exist in three states, BF, BS, and BSS, which interconvert in a cAMP and guanine nucleotide dependent manner. It has been proposed that the transition of BS to BSS represents the activation of a guanine nucleotide regulatory protein [Van Haastert, P.J.M., De Wit, R.J.W., Janssens, P.M.W., Kesbeke, F., & DeGoede, J. (1986) J. Biol. Chem. 261, 9604-9611]. Binding of [3H]cAMP to these sites was measured after a short preincubation with an identical concentration of nonradioactive cAMP. [3H]cAMP could still bind to BF and BS, but not to BSS, indicating that the transition of BS to BSS is blocked by the preincubation with cAMP. This blockade was rapid and showed first-order kinetics with t1/2 = 4 s. A half-maximal blockade was induced by 0.7 nM cAMP; at this concentration only 5% of the B-sites are occupied with cAMP. The blockade of the transition of BS to BSS was released by two conditions: (i) When the concentration of cAMP was increased, the blockade was released within a few seconds. (ii) When cAMP was removed, the blockade was released slowly with t1/2 = 90 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistent cAMP binding by the chemotactic cAMP receptor in the presence of a detergent in Dictyostelium discoideum

We have investigated the effect of a number of detergents on the chemotactic cAMP receptor of Dictyostelium discoideum. 13 detergents were tested; cAMP binding was well preserved only in the presence CHAPS (3[3-cholamidopropyl)dimethylammonio]-1-propanesulphonate) and Zwittergent 3–8 (N-octyl-N,N-dimethyl-3-ammonio-1-propanesulphonate). In the presence of Zwittergent 3–8, cAMP bound to the receptor rapidly exchanged with free cAMP. In contrast, cAMP was persistently bound to the receptor following the addition of CHAPS to membrane-bound receptors pre-equilibrated with cAMP. Binding isotherms indicated that all cAMP-binding sites were similarly affected by CHAPS. The cyclic nucleotide binding specificity of the binding sites that became persistently occupied by cAMP was identical to that of the chemotactic cAMP receptor. Cyclic AMP was not chemically modified by persistent binding. The non-exchanging cAMP-receptor complex was insensitive to modulation by guanine nucleotides and salts such as CaCl₂, MgCl₂, potassium phosphate and ammonium sulphate. We conclude that CHAPS freezes the cAMP-receptor, blocking exchange of free ligand with empty or occupied cAMP-binding sites.     

Ligand-induced modification of a surface cAMP receptor of Dictyostelium discoideum does not require its occupancy

In Dictyostelium discoideum amoebae, cAMP-induced phosphorylation of the surface cAMP receptor is associated with a discrete transition in its electrophoretic mobility. The native and modified forms of the receptor are designated R and D (Mr = 40,000 and 43,000). The relationship of the number of receptors which are modified as a function of the receptors which bind cAMP was investigated. Modification was assessed by determining the amounts of R and D forms in Western blots which detect all receptors whether or not they are exposed on the surface. Cyclic AMP or the analog, adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS), induced a loss of cAMP-binding activity (down-regulation), which was not accompanied by a loss of the receptor protein. About 60% of the receptors do not bind cAMP in the absence of Ca2+ and are unmasked by 10 mM Ca2+. However, the fraction of receptors which are modified in response to cAMP is equal in the absence or presence of Ca2+. (Rp)-cAMPs induces down-regulation (50%) but not modification. Addition of cAMP, following down-regulation by (Rp)-cAMPS, causes all receptors to be modified. cAMP induces both down-regulation (80%) and modification. Modification is more readily reversed than down-regulation: 30 min after removal of cAMP, receptors remain down-regulated (57%) but are found in the R form. All receptors shift to the D form when cAMP is readded to the cells. These results indicate that exposed, as well as cryptic and down-regulated receptors, are modified in response to the cAMP stimulus.     

The cyclic nucleotide specificity of eight cAMP-binding proteins in Dictyostelium discoideum is correlated into three groups

cAMP is a mediator of inter- and intracellular events in Dictyostelium discoideum and is thought to act through specific receptors. Eight forms of cAMP-binding proteins have been described in this organism: four forms of a cell surface receptor, a cell surface and extracellular phosphodiesterase, an intracellular cAMP-dependent protein kinase (CAK), and a recently identified cAMP-binding protein (CABP1) that is present on the cell surface, in the cytoplasm, and in the nucleus. In this study we have analyzed the cyclic nucleotide specificity of these cAMP-binding proteins using 13 derivatives of cAMP with modifications in the adenine, ribose, and phosphate moiety. The results suggest that the cAMP-binding proteins belong to three groups: (i) four forms of the cell surface receptor, (ii) two forms of an intracellular receptor (CABP1 and CAK), and (iii) cell surface and extracellular phosphodiesterase. cAMP is probably bound to the surface receptors in the anti conformation in a hydrophobic cleft of the receptor with essential interactions at N6H2' and O3'. In contrast, cAMP is probably bound to CAK and CABP1 in the syn conformation with essential interactions at O2', O3', O5', and exocyclic oxygen. Finally, binding of cAMP to phosphodiesterase involves only O3' and exocyclic oxygen. The cyclic nucleotide specificity of cAMP-induced processes in D. discoideum indicates that the cell surface receptors participate in the transduction of the cAMP signal during chemotaxis and cell differentiation. Functions for CABP1 and CAK in these processes are presently elusive.     

DC6, a novel type of Dictyostelium discoideum gene regulated by secreted factors but not by cAMP

We have isolated a gene, DC6, which is induced in the early aggregative stages of development in Dictyostelium discoideum. The increase in DC6 expression is dependent on high cell density, indicating that cellular interactions are required for DC6 induction. In low-cell-density cultures, the induction of DC6 occurs if supplied with conditioned medium of developing cells, suggesting that secreted factors are involved in DC6 induction. The expression of DC6 is not affected (1) in the presence of caffeine or adenosine, which block the production or the action of cAMP pulses, (2) in the presence of high concentrations of cAMP, or (3) in mutant strains (Synag7 and FrigidA), which are defective in transduction pathways of cAMP pulse signals. These results indicate that the induction of DC6 does not require extracellular cAMP pulse signals, which are known to regulate the expression of many genes in the early development. Independence of cAMP signals and dependence on other unknown cellular interactions are prominent characteristics of DC6.     

Regulation of guanylate cyclase by a guanine nucleotide binding protein, G alpha 2, in Dictyostelium discoideum.

Binding of cyclic AMP (cAMP) to the cell surface receptor induces a transient activation of guanylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) which lacks G alpha 2, a guanine nucleotide binding protein, does not respond to cAMP. We found that 2,3-dimercapto-1-propanol (BAL) induced a continuous activation both in the frigid and in its parents. Therefore, the BAL-induced continuous activation of guanylate cyclase is independent of G alpha 2. We also found that cAMP enhanced the BAL-induced continuous activation in the frigid mutant. This result suggests that an unidentified signal transduction mechanism from the cAMP-receptor besides the one involving G alpha 2 plays a role in the enhancement of activation. Lastly, we found that the BAL-induced continuous activation was terminated by cAMP in the parental strain, but not in the frigid mutant. Therefore, the cAMP-induced suppression on the BAL-induced continuous activation is mediated through G alpha 2.     

Demonstration of receptor heterogeneity and affinity modulation by nonequilibrium binding experiments. The cell surface cAMP receptor of Dictyostelium discoideum

The binding of [3H]cAMP to Dictyostelium discoideum cells was analyzed on a seconds time scale under both equilibrium and nonequilibrium conditions. The binding of [3H]cAMP increases rapidly to a maximum obtained at about 6 s, which is followed by a decrease to an equilibrium value reached at about 45 s. This decrease of [3H]cAMP binding is not the result of ligand degradation or isotope dilution by cAMP secretion but is due to a transition of high-affinity binding to low-affinity binding. Analysis of the dissociation rate of [3H]cAMP from the binding sites indicates that these high- and low-affinity binding sites are both fast dissociating with a half-life of about 1 s. In addition, these dissociation experiments reveal a third binding type which is slowly dissociating with a half-life of about 15 s. The number and affinity of these slowly dissociating sites does not change during the incubation with [3H]cAMP. The drugs caffeine and chlorpromazine do not change the total number of binding sites, but they change the ratio of the three binding types. In the presence of 10 mM caffeine almost all binding sites are in the low affinity conformation, while in the presence of 0.1 mM chlorpromazine the ratio is shifted to both the high-affinity type and slowly dissociating type. The results indicate that the cAMP-binding activity of D. discoideum cells is heterogeneous. In the absence of cAMP about 4% of the sites are slowly dissociating with Kd = 12.5 nM, about 40% are fast dissociating with high affinity (Kd = 60 nM), and about 60% are fast dissociating with low affinity (Kd = 450 nM). During the binding reaction the number of slowly dissociating sites does not change. The number of high-affinity sites decreases to a minimum of about 10% with a concomitant increase of low-affinity sites to about 90%. This transition of binding types shows first-order kinetics with a half-life of about 9 s. A half-maximal transition is induced by 12.5 nM cAMP.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP-stimulated cGMP phosphodiesterase from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2'H. Modifications at N7, C8, O3' and O5' induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold. Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3', while modifications at N2H2, N3, N7, O2'H, and O5' have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein. cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3-6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.     

Role of a guanine nucleotide binding protein, G alpha 2, in regulation of adenylate cyclase in Dictyostelium discoideum

Two substances, cAMP and 2,3-dimercapto-1-propanol (BAL) are known to induce transient activation of adenylate cyclase in Dictyostelium discoideum. A frigid mutant (HC85) has a deletion in a gene for G alpha 2, a guanine nucleotide binding protein and cannot activate the cyclase in response to cAMP. We found that BAL induced activation in the frigid mutant. This result suggests that the BAL-induced activation is independent of G alpha 2 and that BAL mimics a role of activated G alpha 2. We also found that cAMP promoted the BAL-induced activation. This result suggests that cAMP plays a role in activation through a mechanism in which G alpha 2 is not involved. We lastly showed that continuous cAMP stimulation could not inhibit the BAL-induced activation in the frigid mutant. Since the cAMP-induced inhibition observed in the wild type strain (NC4) proceeds with the time course identical to the cAMP-induced adaptation (Oyama, submitted), this result suggests that G alpha 2 is involved in adaptation of adenylate cyclase.     

Folate and cAMP modulate GTP binding to isolated membranes of Dictyostelium discoideum. Functional coupling between cell surface receptors and G-proteins

In membrane preparations from D. discoideum cells GTP-binding activity is observed. The lack of GTP binding to intact cells suggests that the binding sites are localized inside the cell. The GTP-binding activity also remains in the particulate fraction in the presence of 1 mM Ca++. This excludes membrane-associated microtubuli to be responsible for the observed GTP binding. Scatchard analysis suggests the existence of one type of binding site (Kd = 2.6 microM and 3.6 X 10(5) sites per cell). The kinetics of association as well as dissociation, however, suggest that GTP binding is more complex than binding to a single type of site. GDP and guanylyl imidodiphosphate are potent competitors of GTP binding (respectively 5- and 10-fold worse than GTP) while GMP, cGMP and several adenine nucleotides are ineffective up to 1 mM. The chemoattractants cAMP and folic acid both increase the equilibrium binding level of GTP, while dissociation of GTP is accelerated. These data indicate the functional coupling between cell surface receptors and G-proteins.     

The developmental regulation of L-ornithine decarboxylase in Dictyostelium discoideum and its induction by cAMP

By the use of an in vivo assay, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) is shown to be developmentally regulated in Dictyostelium discoideum. High levels of cAMP can induce ornithine decarboxylase activity in preaggregative cells kept in shaking suspension, under similar conditions as where other markers for development can also be induced. This induction by cAMP is solely dependent on the total amount of cAMP to which the cells have been exposed, and not on the manner of cAMP addition. Induction of ornithine decarboxylase activity, when measured in vitro, is caused by both an increase in total enzyme activity and by a proportional increase in activity of the high-affinity form for the cofactor pyridoxal phosphate. When measured in vivo, an additional regulatory mechanism seems to be involved. Kinetic studies with the competitive inhibitor putrescine suggest that in cAMP-stimulated cells the low affinity form of the enzyme may also be active in vivo.     

cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.     

Kinetics and concentration dependence of reversible cAMP-induced modification of the surface cAMP receptor in Dictyostelium

We have previously reported that extracellular cAMP induced a reversible shift, from apparent Mr = 40,000 to 43,000, in the electrophoretic mobility of a polypeptide identified by photoaffinity labeling with [32P]8-N3-cAMP as the cAMP receptor of Dictyostelium (Klein, P., Theibert, A., Fontana, D., and Devreotes, P. (1985) J. Biol. Chem. 260, 1757-1764). In this report, we examine the kinetics and concentration dependence of this stimulus-induced receptor modification. Prior to stimulation, 90% of the receptors migrated as the higher mobility form (Mr = 40,000) and 10% as the lower mobility form (Mr = 43,000). Following 15 min of persistent stimulation with 1 microM cAMP, the per cent of receptors migrating as the lower mobility form rose to 80%. This transition occurred with a half-time of 2.5 min. Removal of the stimulus initiated a return to the basal state which occurred with a half-time of about 6 min at 22 degrees C. No reversal occurred at 0 degrees C. Addition and removal of a 50 nM cAMP stimulus induced transitions with similar kinetics, but the final plateau value reached was only 40% lower mobility form. The stimulus concentration which induced 50% of the maximal transition from higher to lower mobility forms at steady state was 27 nM, similar to the KD for [3H]cAMP binding. Scatchard analysis of [3H]cAMP binding indicated that, although a 20% down-regulation occurs during cAMP stimulation, there is no significant difference in the affinities of the higher and lower mobility forms of the receptor. The unoccupied higher and lower mobility forms of the receptor, designated R and D, are considered to be in rapid equilibrium with liganded forms, designated RL and DL. The rate constants for interconversion of the receptor forms R (Formula: see text) D and RL (Formula: see text) DL were calculated from the kinetic data: k1 = 0.012, k-1 = 0.104, k2 = 0.222, and k-2 = 0.055. The interconversion steps are not at equilibrium, suggesting that an energy expenditure occurs during the receptor modification. The pattern of modulation of the cAMP-induced receptor modification suggests that it may be the biochemical mechanism of adaptation.     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

Summary We have recently reported the existence of two forms of glycogen phosphorylase (1,4--D-glucan: orthophosphate--glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5AMP and a 104kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 M, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt₂-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 M) for b to a conversion. cDNAs to three cAMP-regulated genes-PL3, Dll, and D3-were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture - a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.Abbreviations EGTA Ethyleneglycol-bis-(-aminoethyl ether) - N,N,N N-tetra acetic acid - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

MYO2 is not essential for viability,but is required for polarized growth and dimorphic switches in Candida albicans

The human fungal pathogen Candida albicans changes from a budding yeast form to a polarized hyphal form in response to various external conditions. Dimorphic switching of C. albicans has been implicated in the development of pathogenicity. Morphogenic transformation requires polarized cell growth and rearrangement of the cytoskeleton. We previously showed that myosins play key roles in the conversion from the bud to the hyphal form of C. albicans by inhibiting myosin activities with 2,3-butanedione-2-monoxime (BDM), a general myosin ATPase inhibitor. In this study we investigated the function of MYO2 in C. albicans using deletion mutants. The amino acid sequence of CaMYO2 shows 60% identity and 77% homology with MYO2 and 54% identity and 70% homology with MYO4 of budding yeast Saccharomyces cerevisiae, suggesting that CaMYO2 is the only class V myosin in C. albicans. Cells in which both CaMYO2 alleles were deleted were viable, suggesting that MYO2 is nonessential in C. albicans. The proliferation of CaMYO2delta cells, however, was sharply decreased. In addition, CaMYO2delta cells showed defects in assembly and polarized localization of F-actin as well as an inability to induce germ tube formation and hyphal growth. The deletion of CaMYO2 also disrupted the shape and migration of the nucleus. These results strongly suggest that CaMYO2 is essential for polarized growth and hyphal transition in C. albicans.