The long physiological reach of the yeast vacuolar H+-ATPase

V-ATPases are structurally conserved and functionally versatile proton pumps found in all eukaryotes. The yeast V-ATPase has emerged as a major model system, in part because yeast mutants lacking V-ATPase subunits (vma mutants) are viable and exhibit a distinctive Vma- phenotype. Yeast vma mutants are present in ordered collections of all non-essential yeast deletion mutants, and a number of additional phenotypes of these mutants have emerged in recent years from genomic screens. This review summarizes the many phenotypes that have been associated with vma mutants through genomic screening. The results suggest that V-ATPase activity is important for an unexpectedly wide range of cellular processes. For example, vma mutants are hypersensitive to multiple forms of oxidative stress, suggesting an antioxidant role for the V-ATPase. Consistent with such a role, vma mutants display oxidative protein damage and elevated levels of reactive oxygen species, even in the absence of an exogenous oxidant. This endogenous oxidative stress does not originate at the electron transport chain, and may be extra-mitochondrial, perhaps linked to defective metal ion homeostasis in the absence of a functional V-ATPase. Taken together, genomic data indicate that the physiological reach of the V-ATPase is much longer than anticipated. Further biochemical and genetic dissection is necessary to distinguish those physiological effects arising directly from the enzyme’s core functions in proton pumping and organelle acidification from those that reflect broader requirements for cellular pH homeostasis or alternative functions of V-ATPase subunits.     

Comparative studies on the physiological ecology of five euryhaline Gammarus species

Summary Comparative investigations on the physiological capacities of Gammarus locusta (L.), G. oceanicus Segerstråle, G. salinus Spooner, G. zaddachi Sexton and G. duebeni duebeni Liljeborg obtained from German coastal and estuarine areas were carried out under uniform experimental conditions. In order to assess the adaptation of these species to the abiotic conditions of their particular habitats, the following criteria were examined: (a) oxygen consumption as related to temperature, (b) time course of acclimation to a new steady state of metabolic rate following sudden temperature changes, (c) resistance to oxygen deficiency, and (d) resistance to aerial exposure. Considerable interspecific differences were found among the five amphipods studied. G. locusta reveals the highest O₂ uptake rates and longest thermal acclimation periods as well as lowest resistance capacities to oxyten depletion and air exposure. The other species occupy an intermediate position as indicated in the above-cited order, but contrast with G. duebeni which exhibits comparatively high resistance capacities. The significance of the findings obtained is discussed in relation to the environmental requirements of the five crustaceans considered.     

干旱胁迫对木兰科5树种生理生化指标的影响

对乐昌含笑(Michelia chapensis Dandy)、金叶含笑(M. foreolate Merr. ex Dandy)、阔瓣含笑(M. platyetala Hand. -Mazz.)、观光木(Tsoongiodendron odorum Chun)和红花木莲[Manglietia insigis (Wall.) Blume]进行28 d干旱胁迫处理,测定了丙二醛(MDA)和可溶性糖含量;对除乐昌含笑以外的4个树种进行了7种脂肪酸成分即肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、α-亚麻酸的测定.结果表明,干旱胁迫时MDA和可溶性糖含量均升高.在4个树种的处理组和对照组中,不饱和脂肪酸比例高于饱和脂肪酸;在干旱胁迫时,饱和脂肪酸比例升高,不饱和脂肪酸比例下降.综合各生理指标变化与田间观察结果,上述5树种按抗旱能力强弱依次排序为:阔瓣含笑、乐昌含笑、观光木、金叶含笑和红花木莲.     

Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase

Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.     

Age-related changes in the physiological state of budding yeast cells

Biochemical and physiological changes occurring during the relative ageing of budding yeast cells were studied. In contrast to scar-free daughter cells the scar-bearing mother cells display a longer G1 phase of the cell cycle, synthesize RNA more slowly, consume oxygen more rapidly, possess a higher saturation constant K_s, and their cell walls are less resistant to mechanical strain.     

Effects of Cd2+ on the physiological state and photosynthetic activity of young barley plants

Barley plants (Hordeum vulgare L. cv. Obzor) were grown as a water culture in a climatic room. One part of them was subjected to a long-term Cd²⁺ stress - 12 d with 5.4×10⁻⁵ M Cd. The Cd²⁺ stress inhibited formation of the photosynthetic apparatus and its capacity for ¹⁴C photoassimilation, decreased the content of soluble proteins, increased the dark respiration rate and the free amino acids content, disturbed plant water relations, as well as the distribution of ¹⁴C within primary photoproducts of the treated barley plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Cd2+ on the physiological state and photosynthetic activity of young barley plants

Barley plants (Hordeum vulgare L. cv. Obzor) were grown as a water culture in a climatic room. One part of them was subjected to a long-term Cd²⁺ stress - 12 d with 5.4×10^?5 M Cd. The Cd²⁺ stress inhibited formation of the photosynthetic apparatus and its capacity for ¹⁴C photoassimilation, decreased the content of soluble proteins, increased the dark respiration rate and the free amino acids content, disturbed plant water relations, as well as the distribution of ¹⁴C within primary photoproducts of the treated barley plants.

     

Comparison of the behavioural and physiological responses to humidity of five species of dry-wood termites, Cryptotermes species

ABSTRACT. A circular humidity gradient apparatus with a porous cellulose substrate was used to compare the humidity responses of pseudoworkers (immature wood-feeding forms) of five species of dry-wood termites. Four of these species are widely distributed pests of buildings: Cryptotermes brevis (Walker), C. cynocephalus Light, C. dudleyi Banks and C. havilandi (Sjöstedt). The fifth species, C. naudei Coaton, has not been recorded from buildings. C. havilandi, C. cynocephalus and C. naudei were found to tend to group and feed at humidites above 90% r.h., while C. dudleyi pseudoworkers avoided humidities above 90% r.h. and below 70% r.h. These results accord with the results of studies on the effects of relative humidity on feeding and reproduction of these species. The behaviour of the most widespread species, C. brevis , seems to be closely associated with its ability to undergo acclimatization and to feed efficiently at either medium ( c. 60%) or high ( c. 90%) relative humidity. C. brevis kept at 89% r.h. showed an increased tendency to group at humidities above 90%, with a lower temperature preference, changes in faecal pellet formation and, for one population at least, an increased tolerance of exposure to saturated air. The humidity preferences at constant temperature were compared with the temperature preferences of the same individuals examined in uniform humidity. The value of behavioural responses as indicators of physiological differences between species and populations is considered.     

Daughter cells as an important factor in determining the physiological state of yeast populations

Cells of Candida utilis grown in a single-stage chemostat at D = 0.05, 0.1, 0.25, and 0.35 hr^?l were separated into a fraction of scar-bearing mother cells and a fraction of scar-free daughter cells. The scar-free cells were transferred into small batch cultures where the length of the maturation phase, changes in length and width of cells, specific growth rate, and specific rate of RNA and protein synthesis were examined for 5 hr. The daughter cells grown at D = 0.05 hr^?1 were very small at the moment of separation from the mother cells (about one-third of the mother cell). Their maturation phase (in a batch culture), at the beginning of which they attain the specific growth rate approaching the μ_max of the strain used, lasts for 3 hr. On the other hand, daughter cells grown at D = 0.35 hr^?1 are almost the same size as the mother cells at the moment of separation. After transfer to a batch culture they begin to bud almost immediately. Similarly, in their other morphological and physiological parameters they differ strikingly from immature daughter cells which are formed at low specific growth rates. The importance of these differences from the point of view of mathematical modeling of growth processes is discussed.     

Effects of a ferrate-containing preparation on diverse metabolic processes in yeast

A plant-sap-derived preparation containing bi-and tervalent ferrate anions was tested on growth, respiration on glucose, and membrane transport of 6-deoxy-d-glucose (6-dGlc) and 2-aminoisobutyric acid (Aib) in several yeast species,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Lodderomyces elongisporus, Rhodotorula gracilis, andDipodascus magnusii. Growth was enhanced by as much as 65%, respiration was not affected significantly except for a decrease inR. gracilis, transport of 6-dGlc was not affected while that of Aib was increased by up to 45% inR. gracilis and up to 27% inL. elongisporus.     

Molecular characterization of the yeast vacuolar H+-ATPase proton pore

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.     

Extinction and colonization processes in subpopulations of five neotropical small mammal species

We report the extinction and colonization rates of five sympatric small mammal species at a semiarid locality in north central Chile. We provide information based on 6 years of monitoring on how colonization and extinction rates change according to landscape features (slope aspect) and on their relationship to populations size, population variability, and body size. We found that: (1) for all species in the assemblage, extinction rates of subpopulations from equatorial-facing slopes were significantly lower than those in polar-facing slopes, (2) population size was the most important factor determining extinction rates, (3) colonization rates did not vary between slopes, and were affected by population size only in equatorial-facing slopes, and (4) most species had higher extinction than colonization rates. Persistence of the metapopulation system for all five small mammal species appears to be fueled by repeated colonization events.     

Evolution of gene families: the multidrug resistance transporter genes in five related yeast species

The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs ("phylogenetic footprints"). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.     

Effects of physiological state on duration of sedation in southern elephant seals

One hundred eighty-one female and thirteen postweanling pup southern elephant seals (Mirounga leonina) were sedated using a combination of ketamine hydrochloride and xylazine hydrochloride. Physiological state had a profound effect on response of the animals to sedation. Physiologically stressed postlactation and postpartum cows had significantly longer periods of sedation than pre-molting females or recently weaned pups. Induction time was not affected by physiological status. Dose rates are recommended for elephant seals in various physiological states.     

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.     

Early steps in assembly of the yeast vacuolar H+-ATPase.

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.     

Genetic and cell biological aspects of the yeast vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase is a member of the third class of H⁺-pumping ATPase. A family of this type of H⁺-ATPase is now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. NineVMA genes that are indispensable for expression of the enzyme activity have been cloned and characterized in the yeastSaccharomyces cerevisiae. This review summarizes currently available information on theVMA genes and cell biological functions of theVMA gene products.     

Monoclonal antibodies against subunits of yeast mitochondrial H+-ATPase

Fourteen stable lines of myeloma-spleen cell hybrids producing antibodies against the mitochondrial H+-ATPase have been isolated. One reacted with the alpha-subunit of the enzyme complex (Mr 56000), nine with the beta-subunit (Mr 54000), and four with a 25 kDa subunit which has not been previously characterized. These antibodies are inhibitory or stimulatory or have no effect upon the enzyme activity. Two of the monoclonal anti-beta-subunit antibodies were found to be particularly effective in immunoprecipitating intact H+-ATPase complex.     

Effects of seed size on dispersal distance in five rodent-dispersed fagaceous species

We studied the effect of seed size on dispersal by comparing dispersal distances in five rodent-dispersed fagaceous species (Lithocarpus harlandii, Quercus variabilis, Q. serrata, Cyclobalanopsis glauca, Castanopsis fargesii) with different seed size. We tracked individual seeds with coded tin-tags in two stands over 3 years in a subtropical evergreen broadleaved forest in the Dujiangyan Region of Sichuan Province, Southwest China. Our seed tracking data indicate that dispersal distances (including mean, maximum and distribution range) of seeds in primary caches and of seeds eaten after dispersal significantly increased with seed size, for both stands and all years. In addition, larger seeds (L. harlandii and Q. variabilis) were re-cached more often than smaller ones, which further reduced the relative density among caches and extended dispersal distances. Our findings indicate that greater dispersal distances for larger seeds might benefit the evolution of differences in seed size, and that scatter-hoarding might be advantageous for rodent-dispersed tree species.     

Dependence of the kinetics of secondary active transports in yeast on H+-ATPase acidification

Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H⁺-ATPase, was inhibited to different degrees by D₂O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide. The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions. An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid. In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mm KC1 had no effect on the transport rates. The ATPase-deficient mutant S. cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and pH were unchanged.Other types of acidification (spontaneous upon suspension; K⁺ stimulated) did not affect the secondary uptake systems.