Electrogenic proton translocation by the ATPase of sugarcane vacuoles

Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation.     

Effects of pressure stress on the fission yeast Schizosaccharomyces pombe cold-sensitive mutant nda3

To investigate the influence of pressure stress on the cell cycle of Schizosaccharomyces pombe, we used a cold-sensitive nda3-KM311 mutant which arrests cell division at a step similar to the mitotic prophase, proposed by Hiraoka and colleagues (Cell 39 (1984) 349-358), under the restrictive temperature, 20 degrees C. The nda3-KM311 cells were first aerobically grown at 30 degrees C, transferred to 20 degrees C for 4 h and shifted to a permissive temperature of 36 degrees C for 15 min. The cells were treated with 100-200 MPa pressure and studied by electron and fluorescence microscopy. At 100 MPa, the nuclear membrane was damaged and the matrix of mitochondria had an electron-dense area. At 150 MPa, the nuclear membrane was broken over broad areas; numerous small vacuoles had fused into large pieces. Actin patches were concentrated in the central region and actin rings were seen in the 20 degrees C-grown cells. Even at 100 MPa, specific actin distribution was lost. Although at 100 MPa, long and fine actin cables were seen all over the cells, large actin patches and the actin rings remained in the center of the cell. They changed into thick and short cables at 150 MPa and above 200 MPa they decomposed but the actin ring was visible even with faint fluorescence. Immunoelectron microscopic observation confirmed this phenomenon.     

Separate Determination of the Electrical Properties of the Tonoplast and the Plasmalemma of the Giant-Celled Alga Valonia utricularis: Vacuolar Perfusion of Turgescent Cells with Nystatin and Other Agents

In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 μm of the H⁺-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to −22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10⁻² to 40.6 × 10⁻²Ωm². The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81–94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 × 10⁻² Fm⁻² compared to 0.77 × 10⁻² Fm⁻² of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system. Received: 22 October 1996/Revised: 16 January 1997     

Ultrastructural localization of ATPase activity in cotton fiber during elongation

Summary The ultrastructural distribution of potassium chloride stimulated adenosine triphosphatase activity was investigated in the outer integument of a linted cultivar of cotton and a lintless (naked seed) mutant from one day preanthesis to eight days postanthesis by using a heavy metal simultaneous capture reaction technique. No enzyme activity other than in mitochondria was observed in the lintless mutant. In the linted cultivar no ATP-specific enzyme activity was seen in non-elongating epidermal cells, subepidermal cells of the outer integuments or any controls. As fiber initials started elongating, enzyme activity gradually appeared on the tonoplasts of enlarging vacuoles. Heavier lead phosphate deposits were observed on the membrane of small vacuoles compared to the tonoplast. This activity continued at least to eight days postanthesis. The enzyme inhibitor, N,N-dicyclohexylcarbodiimide inhibited, while KCl stimulated, tonoplast ATPase activity. The gradual increase of ATPase activity on the tonoplast of expanding fibers, but not on the tonoplasts of non-fiber cells, suggests the active transport of osmotically active compounds, presumably potassium and malate, into the vacuoles of expanding fibers. Fusion of smaller vacuoles with the large central vacuole indicates that these structures contribute additional membrane components along with their enzyme activity to the tonoplast of expanding fibers. The occurrence of ATPase activity, of ER-derived vesicular structures, and the organized pattern of deposition of these structures on the tonoplast indicate ER-originated ATPase activity. This study supports the theory of osmoregulation in cotton fiber where ATPase provides the energy for active accumulation of osmotically active compounds, (K⁺, malate) into the vacuoles, thereby generating and maintaining the turgor pressure required for fiber expansion.Abbreviations ATPase Adenosine triphosphatase - DCCD N,N-Di-cyclohexylcarbodiimide - EM Electron microscope - ER Endoplasmic reticulum - GP -Glycerophosphate - LC Lead citrate - PEP-Case Phosphoenolpyruvate carboxylase - UA Uranyl acetate     

General Mechanisms for Solute Transport Across the Tonoplast of Plant Vacuoles: a Patch-Clamp Survey of Ion Channels and Proton Pumps

The electrical properties of the tonoplast from a large variety of plant materials such as mesophyll cells, storage cells, tumor cells, suspension cultured cells, guard cells, coleoptile cells, and liverwort cells have been investigated using the patch-clamp technique. Whole-vacuole recordings were employed to study the dynamics of an ATP-dependent proton pump by directly measuring the electrogenic currents. The addition of Mg-ATP induced an inwardly directed current which depolarized the tonoplast (the vacuole becoming positive inside). Furthermore, voltage-dependent passive ion fluxes were analyzed using whole vacuoles and isolated membrane patches. Whole-vacuolar currents and single-channel currents were induced at hyperpolarizing potentials, whereas currents decreased at positive trans-tonoplast potentials. The electrical properties of the tonoplast of vacuoles from various plant tissues were similar and it was concluded that ion fluxes across the tonoplast follow the same general mechanisms.     

Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance

Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after long-term culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benz-oxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 × 10⁻⁹ cm² s⁻¹ and a mobile fraction close to 100% at a temperature of 23°C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 × 10⁻⁹ cm² s⁻¹, still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranes—tonoplast and plasma membrane—through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.     

Morphometric study of Lemna gibba in relation to the use of compartmental analysis and the flux-ratio equation in higher plant cells

Morphometric characteristics of Lemna gibba L. cells have been estimated. The mean relative volumes of the wall, of the cytoplasm and of the vacuoles were 0.03, 0.32 and 0.65, respectively. The distribution of the individual values has been studied: for instance 29% of the cells had a mean relative volume of the vacuoles close to 0.73, 39% to 0.68, 19% to 0.61 and 13% to 0.51. The mean value and distribution of the surface areas of the tonoplast and plasmalemma were also determined. This allows us to discuss the active or passive character of the transport of various substances at the plasmalemma and at the tonoplast, according to the usual flux-ratio approach. The feasibility of such an approach in ordinary (non-giant) living cells is discussed with regard to the degree of reliability of the measurements which can be performed with such cells.     

Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica

Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4(+) and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 microM for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.     

Dynamic localization of rop GTPases to the tonoplast during vacuole development

Vacuoles are essential pleomorphic organelles that undergo dynamic changes during cell growth and differentiation in plants. How developmental signals are linked to vacuole biogenesis and development is poorly understood. In this report, we show that a Rop GTPase is localized to developing vacuoles in pea (Pisum sativum cv Extra Early Alaska). Rop belongs to the RHO family of Ras-related small GTP-binding proteins that are key molecular switches in a wide variety of eukaryotic signal transduction pathways. Using indirect immunofluorescence and an anti-Rop antibody, we showed that Rop proteins accumulate to high levels in rapidly growing tapetal cells of pea anthers. In these cells, Rop is localized to an endomembrane system that exists as dynamic pleomorphic networks: a perinuclear fine network decorated with punctate dots, a network composed of small spheres and tubules, and interconnected chambers. Colocalization with a tonoplast annexin VCaB42 shows that these dynamic networks represent the tonoplast. Our results suggest that the dynamic Rop-containing tonoplast networks represent a unique stage of vacuole development. The specific localization of Rop to developing vacuoles supports a role for Rop in signal transduction that mediates vacuole development in plants.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

Sucrose uptake into vacuoles of sugarcane suspension cells

Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H⁺ efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H⁺ antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - Mes 2-(N-morpholino)ethanesulfonic acid - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuriphenylsulfonic acid - PPi pyrophosphate This research was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of H. Schroer is gratefully acknowledged.     

Ultrastructural and cytochemical studies on the developing adhesive disc of Boston Ivy tendrils

Summary Ultrastructural observations of the immature adhesive disc from tendrils of Boston Ivy showed that the peripheral cells, which are the presumptive contact layer, contain vacuoles of varied sizes which are filled with electron-dense aggregates. In small vacuoles, these deposits were appressed to the tonoplast and fusion of these small vacuoles with the large vacuoles apparently occurs. Cells from the central zone were largely parenchymatous. The vacuoles of many of these parenchyma cells contained electron-dense spheres and hemispheres of material either appressed to the tonoplast or within the vacuole lumen. In these cells, the vacuole-cytoplasm interface was characterized by a filiform network of interconnected membranes. Positive reactions with reagents for the identification of polyphenols indicate that the vacuoles of nearly all the peripheral cells and scattered cells of the central zone contain tanniniferous substances. Insoluble carbohydrates also occur in the vacuoles of the peripheral cells, but they contain little or no protein or lipid.     

The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures

Membrane fluidity in whole cells of Saccharomyces cerevisiae W303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6-35 degrees C) and at seven levels of osmotic pressure between 1.38 MPa and 133.1 MPa. An increase in phase transition temperatures was observed with increasing osmotic pressure. At 1.38 MPa, a phase transition temperature of 12 +/- 2 degrees C was observed, which increased to 17 +/- 4 degrees C at 43.7 MPa, 21+/- 7 degrees C at 61.8 MPa, and 24 +/- 9 degrees C at an osmotic pressure of 133.1 MPa. From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range. These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure. Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.     

Multistage disruption of protoplasts by dikegulac

Dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-I-gulonate) is a growth regulator used to differentially kill terminal apices, and it analogously inhibits basic metabolic functions in dividing cells, but not stationary cells, in suspension culture. This report demonstrates an analogous situation in isolated tobacco protoplasts. At the lowest concentrations, dikegulac partially suppresses division of the protoplasts. Higher concentrations are required to produce visual cytoplasmic damage to the protoplasts, which probably first occurs at the level of the plasmalemma, as the vacuoles can be released intact. Later, tonoplast disruption occurs.Abbreviation FDA fluorescein diacetate     

The effect of high temperatures on leaf cells of Valerianella: relative heat stability of the tonoplast membrane of mesophyll vacuoles

The effect of short-term heat stress on the tonoplast membrane of lamb's lettuce (Valerianella locusta (L.) Betcke) mesophyll vacuoles has been investigated. The maintainance of a proton concentration difference (δpH) across the tonoplast membrane served as a criterion for the integrity of the vacuoles. After heat treatment, δpH was measured at room temperature using the fluorescent amine, 9-aminoacridine. It was found with this method that thermal damage to isolated vacuoles mainly occurred in the temperature range above 50°C. Compared with this results, the photosynthetic functions of isolated lettuce protoplasts proved to be markedly more thermolabile, e.g. photosynthetic CO₂ fixation and light-induced chlorophyll fluorescence were drastically reduced at temperatures between 40° and 50°C. Heating of whole leaves and protoplasts and subsequent isolation of vacuoles showed that tonoplast-membrane integrity is not affected by heat stress in situ up to 45°C. Measurement of 9-aminoacridine fluorescence in protoplasts, which allowed conclusions to be drawn regarding the integrity of the tonoplast membrane in its natural cytoplasmic environment, revealed that heat treatment up to 55°C did not significantly affect vacuolar compartmentation. The data provide evidence that the tonoplast membrane is relatively heat stable compared with photosynthetic membranes.     

Ultrastructural Localization of ATPase Activity in the Cells of the Apical Meristem Zone,Elongation Zone and Root Hair Zone of Tomato Roots

A comparative study on the cytochemical localization of adenosine triphosphatase (ATPase) activity reaction in the cells of the apical meristem zone, elongation zone and root hair zone of tomato roots was carried out by electron microscopic observations of lead phosphate precipitation. The following experimental results have been obtained: In the meristematic cells of tomato roots, the heavy lead phosphate deposits indicating a very high activity of ATPase were localized at plasmalemma, plasmodesmata, endoplasmic reticulum, Golgi bodies, nucleoli and chromatin (Figs. 1—2). The reaction products of ATPase activity were also observed at some sites of ground cytoplasm and cell wall, but they were not found in little vacuoles and on tonoplast. In the cells of elongation zone, the ATPase activity at plasmalemma and plasmodesmata was as high as that in the meristematic cells of root tip, while the ATPase activity at nucleoli, chromatin, endoplasmic reticulum and Golgi bodies was markedly lowered. On the other hand, the high ATPase activity was produced on the tonoplast of the developing and enlarging vacuoles (Fig. 3). In the cells of root hair zone, the high ATPase activity was shown at plasmalemma, tonoplast and intercellular spaces, but the ATPase activity at nucleoli, chromatin and endoplasmic reticulum was wholly inactivated. (Figs. 4—7). The above results indicate that the ATPase activity with membranes and organelles is altered when the functions of cells and organelles change. Therefore, it is evident that the ATPase activity may be closely related to many physiological functions.     

The dynamic changes of tonoplasts in guard cells are important for stomatal movement in Vicia faba

Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxy-succinyl-l-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement.     

Malate Uptake into Isolated Vacuoles from Catharanthus roseus Cells: Role of the Membrane Potential

The mechanisms involved in the transport of malate into isolated vacuoles of Catharanthus roseus (L.) cells were investigated with special reference to the effects of induced changes in membrane potential and surface charges of the tonoplast. For this purpose, thiocyanate (SCN^?), a highly permeant anion often used as a membrane potential probe, was extensively exploited. In the absence of Mg-ATP, the low accumulation ratio of ¹⁴C SCN^? could be related to the presence of negative charges at the outer surface of the tonoplast exerting a screening effect on the displacement of lipophilic anionic species. Nevertheless, malate was taken up continuously by vacuoles supporting the concept of a transport component which facilitates its transfer through the tonoplast. From experiments showing the pH dependence of malata uptake, it is suggested that the protonated form of the transporter is implicated in this process. Moreover, when the vacuoles are energized by Mg-ATP, the study of the equilibrium distribution of ¹⁴C SCN^? indicated an inside positive membrane potential difference. Advantage was taken of these results to modulate the membrane potential with high levels of thiocyanate. The data obtained demonstrate that malate uptake results from electrophoretic movement in response to the positive potential difference.     

箭舌豌豆根瘤液泡中细菌周膜来源的研究

电镜观察结果表明,幼龄箭舌豌豆根瘤侵染细胞的细胞质较少,中央是一些体积较大的液泡。细胞质中侵入线经常可见,由侵入线释放出来的细菌均有细菌周膜。这些细菌只位于细胞质中,不出现在液泡里面。成熟根瘤中的侵染细胞与此不同,它们中有大量的成熟侵染细胞,细胞质丰富,里面充满大量细菌,中央常有一个大液泡。当中央液泡发育到一定程度时,位于其附近的细菌可通过液泡膜内吞、液泡膜与细菌周膜融合及液泡膜破裂3种途径进入液泡,后一种途径常伴有寄主细胞质。液泡中的细菌绝大部分裸露在外,只有个别细菌具有细菌周膜且多位于液泡膜的破损处附近,因此细菌周膜可能是原来就有的。     

Lipid phase separations and intramembranous particle movements in the yeast tonoplast

The tonoplast of Saccharomyces cerevisiae contains regions depleted of intramembranous particles as the cells enter stationary phase. Freeze-fracture studies on intact cells from this growth stage show that a dispersed particle distribution predominates if the cell temperature is raised to 40°C but that particle-depleted areas prevail at or below the cell growth temperature of 30°C. Tonoplasts of isolated vacuoles also contain particle-depleted regions. Differential thermal analyses of lipids extracted from isolated vacuoles show an endothermic transition which encompasses the cell growth temperature. These results suggest that the tonoplast at this stage contains patches of gel-phase lipid and that these patches correspond to the intramembranous particle-depleted areas of the freeze-fractured tonoplast.