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PubMed1990

A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 1. Purification and characterization of a novel type of adenosinetriphosphatase that differs from chloroplast F1 adenosinetriphosphatase.

ATPases were solubilized from membranes of Acetabularia acetabulum using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. Three fractions of ATPase, Mono Q-I, -II, and -III, were separated. Activity in fraction Mono Q-I was very labile and could not be accurately determined. Fractions Mono Q-II and -III had specific activities of 0.6 and 6 units/mg of protein, respectively. By SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping, it was shown that fractions Mono Q-II and -III consisted of the same polypeptides with molecular masses of 54K (a-subunit) and 50K (b-subunit). Fractions Mono Q-II and -III had the following catalytic properties: pH optimum at 6.0; substrate specificity, ATP = GTP = ITP much greater than UTP = CTP (Km for ATP 0.6 mM); divalent cation requirement, Mn2+ = Mg2+ greater than Co2+ greater than Zn2+ much greater than Ca2+, Ni2+. Both activities were inhibited by monovalent anions, while monovalent cations had neither inhibitory nor stimulatory effects. Orthovanadate inhibited both activities to 50% at 1 mM, and the most effective inhibitor of both was azide (95% inhibition at 100 microM). An enzyme-phosphate complex was formed after incubation of fraction Mono Q-III with [gamma-32P]ATP. The CF1-ATPase subcomplexes were isolated from the same organism and compared with the fraction Mono Q-III. Data supported the difference of fraction Mono Q-III from CF1-ATPase.

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PubMed1990

A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution.

The Mono Q-III fraction, a Mg2(+)-ATPase, isolated from Acetabularia acetabulum was reconstituted into liposomes of various net charges prepared by the reversed-phase method and tested for a Cl(-)-translocating activity. The liposomes from a mixture of egg lecithin, dicetyl phosphate, and cholesterol (63:18:9 mole ratio, negative liposomes) and from a mixture of egg lecithin and cholesterol (63:9 mole ratio, neutral liposomes) were less leaky than positive liposomes from asolectin, and from a mixture of egg lecithin, stearylamine, and cholesterol (63:18:9 mole ratio). A significant increase in 36Cl- efflux from the negative and neutral liposomes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven 36Cl- efflux was inhibited by addition of azide, an inhibitor of the ATPase. The preincubation of the enzyme with phenylglyoxal, an arginine-modifying reagent, inactivated ATP-mediated 36Cl- efflux, but the ATPase activity of the preparation was not affected. When chloride was replaced by 35SO4(2)-, no ATP-dependent 35SO4(2)- efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed.

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PubMed2001

Expression of V-ATPase proteolipid subunit of Acetabularia acetabulum in a VMA3-deficient strain of Saccharomyces cerevisiae and its complementation study.

The function of the translation products of six different cDNAs for Acetabularia V-ATPase proteolipid subunit (AACEVAPD1 to AACEVAPD6) was examined using a Saccharomyces cerevisiae VMA3-deficient strain that lacked its own gene for one of the proteolipid subunits of V-ATPase. Expression of the cDNAs in the strain revealed that four cDNAs from the six complemented the proton transport activity into the vacuole, visualized by fluorescence microscopy. The vacuolar-membrane-enriched fractions from the four transformants showed cross-reactivity with antibodies against the subunits a and A of S. cerevisiae V-ATPase. Two translation products from the other two cDNAs were demonstrated not to be localized in vacuolar membranes, and thus could not complement the function of the VMA3-deficient strain. As the primary structures deduced from the former four cDNAs are similar but clearly different from those of the latter two, the latter two translation products may not be able to substitute for theVMA3 gene product.

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PubMed1974

Morphology of nucleolar cistrons in a plant cell, Acetabularia mediterranea.

The structural organization of transcriptionally active DNA that contains cistrons for precursor molecules of ribosomal RNA is described in positively stained spread preparations from nuclei and nucleoli isolated from the green alga, Acetabularia mediterranea Lmx. These nuclei contain large aggregates of nucleolar subunits in which fibril-covered regions, the putative active cistrons for precursors of ribosomal RNA, alternate with fibril-free intercepts, the "spacers." The length distribution of the different intercepts of this DNA is given, and the pattern is compared with those shown in animal cell systems. The data are discussed in relation to problems of transcription and of amplification of ribosomal RNA genes.

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PubMed1972

Biosynthesis in isolated Acetabularia chloroplasts. I. Protein amino acids.

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with (14)CO(2) for periods of 6-8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of (14)CO(2) into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with (14)CO(2) in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.

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PubMed1975

Growth of the nuclear envelope in the vegetative phase of the green alga Acetabularia. Evidence for assembly from membrane components synthesized in the cytoplasm.

The primary nucleus of the green alga Acetabularia grows about 25,000-fold in volume while it is separated from the endoplasmic reticulum and the whole cytoplasm by a special paranuclear cisterna of a vacuolar labyrinthum system which shows only very few (two to six per square micrometer) and small (ca. 40-120 nm in diamter) fenestrations. The nuclear envelope does not bear polyribosomes, nor do they occur in the entire zone intermediate between the nuclear envelope and the paranuclear cisterna. It is suggested that this special form of nuclear envelope growth takes place by assembly from cytoplasmically synthesized proteins that are translocated across the paranuclear cisterna in a nonmembrane-structured form.

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PubMed1976

Involvement of bristle coat structures in surface membrane formations and membrane interactions during coenocytotomic cleavage in caps of Acetabularia mediterranea.

In the multinucleate cap rays of the green alga Acetabularia mediterranea the cell surface increases dramatically within a short time period during the final stages of coenocytotomic cleavage. In early stages of cyst formation the cytoplast is traversed by numerous large and prolate cleavage vesicles which are characterized by typical columellar or spinous coat structures. The cleavage vesicles are closely associated with the surface of plastids and, to a lesser degree, of mitochondria. This intimate association seems to be mediated by regularly spaced, densely stained intermembranous cross-bridge structures and is maintained throughout cleavage. These cleavage vesicles contain a finely fibrillar material structurally similar to the hyaline layer of mucilage that fills the space between the plasma membrane and cell wall. They line up with invaginations of the plasmalemma and vacuole membranes and, together with smaller vesicles interspersed, constitute preformed "perforation lines" for the final separation of the coenoblast portions. Equidistantly spaced plaques of attachment of such vesicles with surface membrane are described. We hypothesize (a) that the cleavage vesicle membrane is the immediate precursor to the new postcoenocytotomic surface membrane, (b) that the cleavage vesicle coat structures are integrated into the subsurface coat of the plasma membrane, (c) that growth of the laterally attached cleavage vesicles by intussusception of small fuzzy-coated vesicles is confined to their "free ends," (d) that the intermembranous cross-bridge elements are related to bristle coat structures and play a role in the establishment of the cleavage lines, and (e) that the coenocytotomic cleavage process is organized so that adjacent plastids are separated in a way that guarantees the inclusion of several plastids in each cyst.

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PubMed1976

Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane.

The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.

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PubMed2006

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum.

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.

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PubMed2002

Differential messenger RNA gradients in the unicellular alga Acetabularia acetabulum. Role of the cytoskeleton.

The unicellular green alga Acetabularia acetabulum has proven itself to be a superior model for studies of morphogenesis because of its large size and distinctive polar morphology. The giant cell forms an elongated tube (a stalk of up to 60 mm in length), which at its apical pole makes whorls of hairs, followed by one whorl of gametophores in the shape of a cap. At its basal pole, the cell extends into a rhizoid wherein the single nucleus is positioned. In this study, we have determined the level of specific messenger RNAs in the apical, middle, and basal regions using reverse transcriptase-PCR methodology. Four mRNA classes were distinguished: those that were uniformly distributed (small subunit of Rubisco, actin-1, ADP-glucose, centrin, and alpha- and beta-tubulin), those that expressed apical/basal (calmodulin-4) or basal/apical gradients (calmodulin-2 and a Ran-G protein), and those with development-specific patterns of distribution (mitogen-activated protein kinase, actin-2, and UDP-glucose-epimerase). Restoration of the apical/basal calmodulin-4 mRNA gradient after amputation of the apical region of the cell requires the nucleus and was abolished by cytochalasin D. Accumulation of actin-1 mRNA in the vicinity of the wound set by the amputation needs, likewise, the presence of the nucleus and was also inhibited by cytochalasin. This suggests that actin microfilaments of the cytoskeleton are involved in directed transport and/or anchoring of these mRNAs.

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PubMed2001

Asymmetric subcellular mRNA distribution correlates with carbonic anhydrase activity in Acetabularia acetabulum.

The unicellular green macroalga Acetabularia acetabulum L. Silva is an excellent system for studying regional differentiation within a single cell. In late adults, physiologically mediated extracellular alkalinity varies along the long axis of the alga with extracellular pH more alkaline along the apical and middle regions of the stalk than at and near the rhizoid. Respiration also varies with greater respiration at and near the rhizoid than along the stalk. We hypothesized that the apical and middle regions of the stalk require greater carbonic anhydrase (CA) activity to facilitate inorganic carbon uptake for photosynthesis. Treatment of algae with the CA inhibitors acetazolamide and ethoxyzolamide decreased photosynthetic oxygen evolution along the stalk but not at the rhizoid, indicating that CA facilitates inorganic carbon uptake in the apical portions of the alga. To examine the distribution of enzymatic activity within the alga, individuals were dissected into apical, middle, and basal tissue pools and assayed for both total and external CA activity. CA activity was greatest in the apical portions. We cloned two CA genes (AaCA1 and AaCA2). Northern analysis demonstrated that both genes are expressed throughout much of the life cycle of A. acetabulum. AaCA1 mRNA first appears in early adults. AaCA2 mRNA appears in juveniles. The AaCA1 and AaCA2 mRNAs are distributed asymmetrically in late adults with highest levels of each in the apical portion of the alga. mRNA localization and enzyme activity patterns correlate for AaCA1 and AaCA2, indicating that mRNA localization is one mechanism underlying regional differentiation in A. acetabulum.

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PubMed2005

Development and organization of the central vacuole of Acetabularia acetabulum.

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.

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PubMed1991

A re-examination of the role of the nucleus in generating the circadian rhythm in Acetabularia.

The role of the nucleus in the generation of the circadian rhythm in Acetabularia has been nuclear. Early experiments showed that the plant could exhibit a circadian rhythm in the absence of a nucleus. However, other experiments appeared to show that the nucleus could impart phase information to the rhythm, and so therefore must be a part of the system that generates the rhythm. We have conducted experiments similar to these--in particular, one in which the nuclear end of the plant was entrained on a light-dark cycle that was opposite that of the rest of the plant. The phase of the free-running rhythm of this type of plant is not consistent with the conclusion that the nucleus is part of the circadian oscillator. We have also tried entraining opposite ends of plants with no nuclei on opposite light-dark cycles. The ultimate phases of these plants appear to be nearly random. A possible interpretation of these experiments is discussed.

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PubMed1979

Accumulation of peroxidase in the cap rays of Acetabularia during the development of gametangia.

Accumulation of peroxidase was demonstrated by light and electron microscopy to occur in Acetabularia in certain regions of the cap rays in relation to the development of the gametangia (cysts). Peroxidase was found to be incorporated into special, cell wall-like obstructions that separate the cap rays from the stalk when the secondary nuclei have settled in the cap rays. It is assumed that peroxidase acts as an anti-microbial protectant of the gametangia.

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PubMed2004

Comparison of ESTs from juvenile and adult phases of the giant unicellular green alga Acetabularia acetabulum.

BACKGROUND: Acetabularia acetabulum is a giant unicellular green alga whose size and complex life cycle make it an attractive model for understanding morphogenesis and subcellular compartmentalization. The life cycle of this marine unicell is composed of several developmental phases. Juvenile and adult phases are temporally sequential but physiologically and morphologically distinct. To identify genes specific to juvenile and adult phases, we created two subtracted cDNA libraries, one adult-specific and one juvenile-specific, and analyzed 941 randomly chosen ESTs from them. RESULTS: Clustering analysis suggests virtually no overlap between the two libraries. Preliminary expression data also suggests that we were successful at isolating transcripts differentially expressed between the two developmental phases and that many transcripts are specific to one phase or the other. Comparison of our EST sequences against publicly available sequence databases indicates that ESTs from the adult and the juvenile libraries partition into different functional classes. Three conserved sequence elements were common to several of the ESTs and were also found within the genomic sequence of the carbonic anhydrase1 gene from A. acetabulum. To date, these conserved elements are specific to A. acetabulum. CONCLUSIONS: Our data provide strong evidence that adult and juvenile phases in A. acetabulum vary significantly in gene expression. We discuss their possible roles in cell growth and morphogenesis as well as in phase change. We also discuss the potential role of the conserved elements found within the EST sequences in post-transcriptional regulation, particularly mRNA localization and/or stability.

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PubMed1975

Cytoplasmic induction of changes in the ultrastructure of the Acetabularia nucleus and perinuclear cytoplasm.

The ultrasturcture of the cell nucleus and perinuclear cytoplasm in Acetabularia is quite different in young and old cells. When a nucleus of an old cell was implanted into the cytoplasm of a young cell the nucleus assumed the morphology typical of a young cell within less than 10 days. The cytoplasm of an old cell was able to induce the reverse change in an implanted nucleus from a young cell. This cytoplasmic induction of nuclear ageing appeared to proceed more rapidly than rejuvenation.

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PubMed1983

Thymidylate kinase from Acetabularia. II. Regulation during the life cycle.

The activity of dTMP kinase (EC 2.7.4.9) was estimated during the development of Acetabularia. Enzyme activity was increased at the beginning of the generative phase. Regulation of the dTMP kinase activity was observed even in the absence of the nucleus. More than 30 days after enucleation the enzyme activity increased two- to threefold. The increase in activity was inhibited by puromycin and cycloheximide but not by rifampicin and chloramphenicol. These results indicate that the enzyme is coded by the nuclear genome and translated on 80 S ribosomes. From mixing experiments with low-activity and high-activity cell extracts it is concluded that the regulation is due to de novo synthesis of the enzyme.

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PubMed1985

Regulation of a ribonucleoside reductase during the early generative phase in Acetabularia.

The activity of a ribonucleoside reductase was estimated during the life cycle of Acetabularia. During the early generative phase the enzyme activity was dramatically increased. Regulation of the ribonucleoside reductase was observed even in the absence of the nucleus. The increase in activity was inhibited by chloramphenicol but not by cycloheximide. These results indicate that the enzyme is translated on 70 S ribosomes.

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