Of proteins and RNA: The RNase P/MRP family

Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

Accumulation of unsulphated precursors inDictyostelium discoideum during selenate inhibition of growth

Incubation ofDictyostelium discoideum cells with selenate is known to inhibit vegetative growth. In this paper we show that in the presence of selenate macromolecules accumulate which can be converted to sulphated products once the selenate is removed. The presence of cycloheximide, an inhibitor of protein synthesis, during the subsequent incubation does not prevent this conversion but tunicamycin, an inhibitor of glycosylation does. It is concluded that, in the presence of selenate, precursors accumulate as unglycosylated proteins, suggesting that feedback inhibition of glycosylation may be operated.     

In vitro selection of an archaeal RNase P RNA mimics natural variation

Archaeal and bacterial RNase P RNAs are similar in sequence and secondary structure, but in the absence of protein, the archaeal RNAs are much less active and require extreme ionic conditions for activity. To assess how readily the activity of the archaeal RNA alone could be improved by small changes in sequence, in vitro selection was used to generate variants of a Methanobacterium formicicum RNase P RNA: Bacillus subtilus pre-tRNA(Asp) self-cleaving conjugate RNA. Functional variants were generated with a spectrum of mutations that were predominately consistent with natural variation in this RNA. Variants generated from the selection had cleavage rates comparable to that of wild type; variants with improved cleavage rates or lower ionic requirements were not obtained. This suggests that the RNase P RNAs of Bacteria and Archaea are globally optimized and the basis for the large biochemical differences between these two types of RNase P RNA is distributed in the molecule.     

Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

Analysis of the tertiary structure of bacterial RNase P RNA

The ubiquitous occurrence of ribonuclease P (RNase P) as a ribonucleoprotein and the catalytic properties of bacterial RNase P RNAs indicate that RNA fulfills an ancient and important role in the function of this enzyme. This review focuses on efforts to determine the structure of the bacterial RNase P RNA ribozyme. Phylogenetic comparative analysis of a library of bacterial RNase P RNA sequences has resulted in a well-developed secondary structure model and allowed identification of some elements of tertiary structure. The native structure has been redesigned by circular permutation to facilitate intra- and inter-molecular crosslinking experiments in order to gain further structural information. The crosslinking constraints, together with the constraints provided by comparative analyses, have been incorporated into a first-order model of the structure of the ribozyme-substrate complex. The developing structural perspective allows the design of self-cleaving pre-tRNA-RNase P RNA conjugates which are useful tools for additional structure-probing experiments.Abbreviations cpRNA circularly permuted RNA     

Environmental factors inducing sexual development inDictyostelium discoideum

In the heterothallic strains NC4 and HM1 ofDictyostelium discoideum, sexual development is initiated by the formation of diploid zygotic giant cells produced through the fusion of these two opposite mating-type haploid cells. For sexual cell fusion, amoeboid cells must first acquire fusion competence, which requires culture under certain environmental conditions, such as darkness, excessive water, and sufficient bacteria as food. However, in the subsequent stages of cell fusion and development of the giant cells into mature macrocysts, cells do not require the above conditions. Cell fusion and development into macrocysts were able to occur even in light with minimum water and in the absence of bacteria. For cell fusion calcium ions were required.     

The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P

Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.     

Spore germination in Dictyostelium discoideum

Mutant spores of Dictyostelium discoideum, strain SG-10, differ from wild type spores in their ability to spontaneously germinate, to be activated with 5% dimethyl Sulfoxide (DMSO), and to be deactivated with 0.2 M sucrose. Both heat-activated wild type and mutant spores began to swell after a lag of 60–75 min at ambient temperature. Suspension of heat activated spores in 5% DMSO resulted in blockage of spore swelling and a concomitant severe inhibition of respiration; removal of 5% DMSO allowed resumption of respiration and the spores began to swell after a lag of only 15 min. It was concluded that 5% DMSO allowed the early reactions (M) to proceed but blocked the later reactions (R) of post-activation lag.Treatment of one day old spores with 20% DMSO solution for 30–120 min quantitatively activated the population. The post-activation lag time was directly dependent on the time of 20% DMSO treatment. Spores activated with 20% DMSO treatment could be deactivated by incubation at 0°C; the spores most quickly deactivated at 0°C were those within 10 min of swelling. Mitochondrial transport inhibitors such as azide and cyanide caused deactivation in an analogous manner. It is hypothesized that spores proceed to the second portion of the lag phase called (R) before the environment determines if dormancy is reimposed or if germination will proceed. The sensitive strain (SG-10) showed a greater degree of damage than the wild type after supraoptimal treatment with 40% DMSO. The spores became more resistant with age to the damaging action of 40% DMSO. All the observed effects of DMSO treatment were compatible with our multistate model of activation which suggests that the early portion of the lag phase (M) may involve a relative uncoupling of oxidative phosphorylation while the later portion (R) may require tight coupling.     

Expression of starvation-induced genes in zygotes ofDictyostelium discoideum

Two cDNA fragments induced in developing zygotes ofDictyostelium discoideum were isolated by mRNA differential display. the relevant genes were also found to be expressed during asexual development, suggesting that sexual and asexual development share common molecular mechanisms inD. discoideum.     

Periodic stimuli are more successful than randomly spaced ones for inducing development inDictyostelium discoideum

Aggregation in the cellular slime moldDictyostelium discoideum is due to chemotaxis. The chemoattractant, cyclic AMP, is synthesised and released periodically by the cells. Externally applied periodic pulses of cyclic AMP can also induce differentiation in this organism. The present work examines the role of periodicityper se in cyclic AMP-mediated stimulation of cell differentiation. For this purpose we use Agip53, aDictyostelium mutant which does not develop beyond the vegetative state but can be made to aggregate and differentiate by reiterated applications of cyclic AMP. Importantly, Agip53 cells do not make or release any cyclic AMP themselves even in response to an increase in extracellular cyclic AMP. A comparison of the relative efficiencies of periodic and aperiodic stimulation shows that whereas the two patterns of stimulation are equally effective in inducing the formation of EDTA-stable cell contacts, periodic stimuli are significantly superior for inducing terminal differentiation. This suggests that there must be molecular pathways which can only function when stimulation occurs at regular intervals.     

Glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds Dictyostelium discoideum and D. mucoroides

Six monoclonal antibodies were isolated which react with common antigens shared by multiple glycoconjugate species in the cellular slime mold Dictyostelium discoideum. Based on competition of antibody binding by glycopeptides and simple sugars, and inhibition of antibody binding by antigen pretreatment with Na periodate, it is argued that at least five of the six antibodies recognize epitopes which contain carbohydrate. These epitopes are consequently referred to as glycoantigens (GAs).Three of the GAs are expressed during growth and throughout the developmental cycle, but are eventually enriched in prestalk and stalk cells. The remaining three are expressed only during and/or after aggregation and are exclusively expressed or highly enriched in prespore cells and spores. These conclusions are derived from Western blot immunoanalysis of purified cell types, immunofluorescence, and EM immunocytochemistry.The two GAs found only in prespore cells appear to be exclusively enclosed within prespore vesicles. The third GA of this type, which is only enriched in prespore cells compared to prestalk cells, is also found in other vesicle types as well as on the cell surface.Two of the GAs enriched in prestalk cells are initially found in all cells of the slug. They are undetectable in spores and prominent in stalk cells. The third GA, though found in the interiors of both prestalk and prespore cells, is enriched on the cell surface of prestalk cells.The chief characteristics of expression of four of these GAs are conserved in the related species D. mucoroides. This species is characterized by continuous trans differentiation of prespore cells into prestalk cells. This shows that the prespore cells maintain specific mechanisms for turning over their cell type specific GAs and that prestalk cells express a specific mechanism for inducing at least one of their cell-type specific GAs.These observations identify specific carbohydrate structures (as GAs) whose synthesis, subsequent localization and turnover are developmentally regulated. The exclusive association of two GAs with prespore vesicles identifies these GAs as markers for this organelle and raises questions regarding the functional significance of this association. The restricted cell surface localization of the other four GAs, together with data from cell adhesion studies, suggest the possibility of a potential role for these GAs in intercellular recognition leading to cell sorting.This paper is dedicated to the memory of the late Daniel McMahon.     

Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum

Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis–Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.     

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum

The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated DdKif3-592 motor (aa 1-592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0micrcom s(-1) at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1-342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFP-fusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than animal kinesins.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors inDictyostelium discoideum

Summary Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolatedDictyostelium discoideum membranes. The K_0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 x 10⁻⁴, 1.3 X 10⁻², and higher than 0.1 s⁻¹. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (K_D is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal inD. discoideum.     

Development of an autophagic system in differentiating cells of the cellular slime moldDictyostelium discoideum

Summary Changes in an autophagic system during differentiation of cells ofDictyostelium discoideum, NC-4 were studied under light and electron microscopes, and it was demonstrated cytochemically that acid phosphatase was almost exclusively localized in food and autophagic vacuoles. Autophagic vacuoles first appeared during formation of loose aggregates, coupled with the defecation of food vacuoles. Autophagic vacuoles seem to originate from flat sacs which segregate parts of the cytoplasm. No acid phosphatase was detected in the vacuoles when first formed, but activity appeared later probably due to fusion with Golgi-like vesicles. When starved cells were not allowed to aggregate due to a low cell density, they formed no autophagic vacuoles but retained many food vacuoles. This indicates that the formation of autophagic vacuoles is not simply due to starvation, but to cell interaction mediated by cell contact. Autophagic vacuoles containing acid phosphatase rapidly increased in number in all cells in the early stage of aggregation. After papillae formed, however, they selectively decreased in the prespore cells, but developed further and grew larger in the prestalk cells.