Structure and function of the yeast vacuolar membrane proton ATPase

Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H⁺-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F₀F₁-type ATP synthase and the plasma membrane E₁E₂-type H⁺-ATPase. The vacuolar membrane H⁺-ATPase is composed of three major subunits, subunita (M _r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H⁺-ATPase under two kinetic conditions have been determined to be 0.9–1.1×10⁵ daltons for single-cycle hydrolysis of ATP and 4.1–5.3×10⁵ daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H⁺-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP carbonyl cyanidem-chlorophenyl hydrazone - DCCD N,N-dicyclohexylcarbondiimide - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Pi inorganic phosphate - SDS sodium dodecylsulfate - SF6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile - SITS 4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid - ZW3-14 N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate     

The adverse effects of HEPES,TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

Summary Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO₂ enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO₂ enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 μm diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution. This work forms part of a project on Connective Tissue Remodelling supported and financed by the Medical Research Council of New Zealand, of which M. H. F. is a Career Fellow.     

Recycling of mast cells following degranulation in vitro: An ultrastructural study

Mature mast cells, isolated from the rat peritoneal cavity, were placed into suspension culture, either as resting cells or after degranulation by exposure to compound $\text{48}\text{80}$, and were maintained for up to 63 hr. No mitotic cells were observed, and cell number was conserved. The culture conditions did not cause spontaneous degranulation and cell survival was better than 80%. However, with time in culture, an increasing percentage of cells acquired a vesiculated appearance, characterized by a Golgi area with distended cisternae, the accumulation of lysosomal or autophagic-like vesicles, and enlarged, irregular or fused secretory granules. In the degranulated group, about one-fourth of the cells recovered the morphological appearance of resting cells by 63 hr, indicating that they are capable of ‘recycling’. A cell type with a unique morphology, characterized by a large central vacuole containing secretory product, an eccentric nucleus, and mature secretory granules at the cell periphery appeared in the stimulated group after 22 hr of culture. It may be a possible intermediate stage in the mast cell regranulation process, based on its occurrence exclusively in the stimulated group, the correlation between its distribution and the recovery of mast cells to the resting state, and the morphological resemblance of its granule contents to stages in granule maturation in differentiating embryonic mast cells.     

Morphological and histochemical study of the endometrial effects of Ovral in the baboon

Summary In an effort to better understand changes induced by hormonal contraceptives, a group of female baboons were administered Ovral for a period of 9 months. During this time the endometrium was sampled by transcervical uterine biopsy from both the treated animals and from a control group. The biopsies were all obtained between 10 and 14 days of the treatment cycle or the normal menstrual cycle. The endometrial glandular cells from the treated animals exhibited an accelerated maturation compared with the controls. Ultrastructurally this was reflected by increased cell size, numerous long, slender microvilli on the apical membranes, and increased development of the Golgi complex. Differences were also observed in the predominant type of granule seen in the apical cytoplasm. After 3 and 6 months of treatment with Ovral, no significant differences were noted between groups or between animals within a group. However, after 9 months of treatment, the endometrium displayed differences from the earlier experimental groups as well as individual variations. The functional correlates of these observations are discussed and compared to human endometrium.     

Sphingomyelinase Treatment Induces ATP-independent Endocytosis

ATP hydrolysis has been regarded as a general requirement for internalization processes in mammalian cells. We found, however, that treatment of ATP-depleted macrophages and fibroblasts with exogenous sphingomyelinase (SMase) rapidly induces formation of numerous vesicles that pinch off from the plasma membrane; the process is complete within 10 min after adding SMase. By electron microscopy, the SMase-induced vesicles are ~400 nm in diameter and lack discernible coats. 15–30% of plasma membrane is internalized by SMase treatment, and there is no detectable enrichment of either clathrin or caveolin in these vesicles. When ATP is restored to the cells, the SMase-induced vesicles are able to deliver fluid-phase markers to late endosomes/lysosomes and return recycling receptors, such as transferrin receptors, back to the plasma membrane. We speculate that hydrolysis of sphingomyelin on the plasma membrane causes inward curvature and subsequent fusion to form sealed vesicles. Many cell types express a SMase that can be secreted or delivered to endosomes and lysosomes. The hydrolysis of sphingomyelin by these enzymes is activated by several signaling pathways, and this may lead to formation of vesicles by the process described here.     

Photosynthesis dependent acidification of perialgal vacuoles in theParamedum bursaria/Chlorella symbiosis: Visualization by monensin

Summary After treatment with the carboxylic ionophore monensin theChlorella containing perialgal vacuoles of the greenParamecium bursaria swell. TheParamecium cells remain motile at this concentration for at least one day. The swelling is only observed in illuminated cells and can be inhibited by DCMU. We assume that during photosynthesis the perialgal vacuoles are acidified and that monensin exchanges H⁺ ions against monovalent cations (here K⁺). In consequence the osmotic value of the vacuoles increases. The proton gradient is believed to drive the transport of maltose from the symbiont into the host. Another but light independent effect of the monensin treatment is the swelling of peripheral alveoles of the ciliates, likewise indicating that the alveolar membrane contains an active proton pump.Abbreviations HEPES N-(2-hydroxyethyl)piperazine-N-2-ethane sulfonic acid - DCMU 3-(3, 4-dichlorophenyl)-1,1-dimethylurea     

Microbial ecology of planktonic filamentous phototrophic bacteria in holomictic freshwater lakes

The microbial ecology of the filamentous phototrophic bacterium, Chloronema giganteum, has been studied in the water column of three central European lakes (Schlein, Buchen and Vechten). In these lakes an anoxic layer, termed the transition zone, was located between the oxycline and the redoxcline. The migration capacity of Chloronema through this zone appears to be responsible for the natural preponderance of either straight or spiral forms. When the transition zone is less than 1 m thick the straight form is dominant, but when this transition zone is wider than 2 m the spiral form is enriched. The intermediate situations favour both filamentous forms.     

PEG-enhanced,electric field-induced fusion of tonoplast and plasmalemma of grape protoplasts

Cultured grape cells accumulate anthocyanins in vacuoles rather than secreting them into the nutrient medium. Therefore, grape cells that contain tonoplast segments in their plasmalemma should be capable of excreting anthocyanins rather than sequestering them in their vacuoles. In initial attempts to construct such novel cells, small vacuoles were fused with the plasmalemma of cultured plant cells. Protoplasts were isolated from grape calluses that produce and accumulate anthocyanins. Small vacuoles were formed by gently rupturing vacuoles isolated from grape protoplasts. Although small vacuoles and protoplasts became aligned in an AC field, the tonoplast and plasmalemma did not readily fuse when subjected to 3 DC pulses of 1200 V cm^–1 for 50 s each. Changes in the intensity, number and/or duration of the DC pulses had no effect on the fusion process. When 1.0% polyethylene glycol was added to the electrofusion buffer, however, small vacuoles and protoplasts fused within a few minutes after the DC pulses were applied. These novel grape cells remained viable for several hours.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichloro-phenoxyacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-amino-ethyl ether)-N,N,N,N - MES 2-[N-Morpholino]ethanesulfonic acid - MOPS 3-[N-Morpholino]propanesulfonic acid - PVP polyvinylpyrrolidone     

Fusion of Host Cell Secondary Lysosomes with the Parasitophorous Vacuoles of Leishmania mexicana-inlected Macrophages

SYNOPSIS. Secondary lysosomes of cultured mouse peritoneal macrophages were labeled with the electron-dense colloid saccharated iron oxide; the identity of the labeled structures was checked by the Gomori reaction for acid phosphatase. Amastigotes of Leishmania mexicana mexicana derived from mouse lesions were used to infect these macrophages in vitro. In electron micrographs of thin sections of infected macrophages the labeled secondary lysosomes were seen fused with the parasitophorous vacuoles without preventing subsequent multiplication of the parasites. A similar fusion probably occurs in vivo , and may provide a pathway through which not only nutrients but also drugs and host antibodies could reach the intracellular parasite.     

Autophagie in den Drüsenzellen des Magens bei jungen Mäusen

Zusammenfassung Autophagische Prozesse in verschiedenen Drüsenzellen des Magens neugeborener bis 8 Tage alter Mäuse wurden elektronenmikroskopisch untersucht. Es zeigte sich dabei, daß die intrazellulären Abbauprozesse in jeder Zellart typisch verlaufen. In den mukoiden Nebenzellen findet man große autophagische Vakuolen mit zahlreichen Mitochondrien, in denen beträchtliche Cytoplasmaregionen sequestriert sein können. Stärker abgebaute Mitochondrien liegen oft im Drüsenlumen oder in den interzellulären Räumen. Anscheinend wird der Inhalt autophagischer Vakuolen in sie ausgeschieden. Alle Zellen mit Autophagosomen enthalten gleichzeitig viele und formenreiche Lysosomen. Einige abgenutzte Zellen des mukoiden Oberflächenepithels scheinen durch Heterophagie abgebaut zu werden. In kleinen Autophagosomen dieser Zellen findet man nur einzelne Organellen. In den Belegzellen ist die Autophagie viel seltener. Typisch für die Belegzellen sind stark osmiophile multivesikuläre Körperchen, die sehr wahrscheinlich Lysosomen verkörpern. In den Hauptzellen, die erst nach der Geburt ausdifferenziert werden, trifft man nur ausnahmsweise sequestrierte Zellpartien.Die Ergebnisse werden unter Berücksichtigung von Beobachtungen anderer Autoren, die die intrazellulären Abbauprozesse analysierten, und eigener früher erzielter Resultate diskutiert.

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Autophagy in gastric gland cells of young mice

Summary Autophagy in different gastric gland cells of newborn till 8 days old mice were studied electronmicroscopically. Intracellular digestion goes on in a way typical for each cell type. In the mucous neck cells, big autophagic vacuoles occur in which a large part of cytoplasm with numerous mitochondria is separated. The damaged mitochondria lie very often in the gland lumen or in the intercellular space. It seems, that the contents of autophagic vacuoles can be extruded in these places. All cells with autophagic vacuoles contain also numerous lysosomes of different shape. Some involuted mucous cells of the surface epithelium seem to be digested in a heterophagic process. Autophagy does not appear often in parietal gastric cells. The autophagosomes of these cells are small, and they contain only some organelles. Typical for these elements are osmiophilic multivesicular bodies, probably lysosomes. Chief cells, that differentiate only after birth, have autophagic vacuoles, but seldom. The results are discussed in comparison with results of other authors that worked on the problems of intracellular digestion.

Der Jugoslawischen Forschungsgemeinschaft Savezni Fond (Belgrad) und Sklad Borisa Kidria (Ljubljana) danken wir für finanzielle Unterstützung, Frau B. Zorman und Herrn F. Kovai für technische Hilfe.