Endocytosis,digestive vacuolar movement and exocytosis on refeeding starvedTetrahymena pyriformis GL-9

Summary Evidence is presented in support of the hypothesis that the contents of digestive vacuoles in refed starvedTetrahymena pyriformis GL-9 are egested from the cell in approximately the sequence of their order of formation. The investigations involved measurements of the rates of disappearance of digestive vacuoles from the cells and the subsequent appearance of egested globules in the surrounding medium using both cultures and individual cells. The cells were first fed peptone and latex particles for a period and then this type of vacuole formation was suppressed by the addition of excess carmine particles (or the process was repeated with the particles in reverse order). Thus two types of morphologically distinct digestive vacuoles could be produced and observed microscopically. These observations suggest that the temporal nature of the movement of the digestive vacuoles through the cell result in the temporal nature of egestion and that no selective mechanism occurs at egestion. Thus digestive vacuoles are thought to pass through the cell from cytopharynx to cytoproct in approximately the order formed and at approximately constant rate. Under conditions of excess nutrients, where the cells become filled with digestive vacuoles, they seem to be able to maintain an approximately uniform number of digestive vacuoles within themselves by maintaining approximately constant and equal rates of vacuole formation and egestion. The maximum rates of latex or carmine vacuole formation or egestion found in single cells were approximately 0.3–0.4 vacuoles per cell per minute. The results are discussed.     

Sustained Food Vacuole Formation by Axenic Paramecium tetraurelia and the Inhibition of Membrane Recycling by Alcian Blue

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.     

Sustained food vacuole formation by axenic Paramecium tetraurelia and the inhibition of membrane recycling by Alcian Blue.

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.     

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

Summary Although it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t_1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.Abbreviations BS-FITC bovine serum albumin-fluorescein isothiocyanate - CVC contractile vacuole complex - DV-I to DV-IV digestive vacuole stages 1 to 4 - HRP horseradish peroxidase - V-ATPase vacuolar proton pump     

Mechanistic differences in the uptake of salicylic acid glucose conjugates by vacuolar membrane‐enriched vesicles isolated from Arabidopsis thaliana

Salicylic acid (SA) is a plant hormone involved in a number of physiological responses including both local and systemic resistance of plants to pathogens. In Arabidopsis, SA is glucosylated to form either SA 2‐O‐β‐d ‐glucose (SAG) or SA glucose ester (SGE). In this study, we show that SAG accumulates in the vacuole of Arabidopsis, while the majority of SGE was located outside the vacuole. The uptake of SAG by vacuolar membrane‐enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate (ABC transporter inhibitor) and bafilomycin A₁ (vacuolar H⁺‐ATPase inhibitor), suggesting that SAG uptake involves both an ABC transporter and H⁺‐antiporter. Despite its absence in the vacuole, we observed the MgATP‐dependent uptake of SGE by Arabidopsis vacuolar membrane‐enriched vesicles. SGE uptake was not inhibited by vanadate but was inhibited by bafilomycin A₁ and gramicidin D providing evidence that uptake was dependent on an H⁺‐antiporter. The uptake of both SAG and SGE was also inhibited by quercetin and verapamil (two known inhibitors of multidrug efflux pumps) and salicin and arbutin. MgATP‐dependent SAG and SGE uptake exhibited Michaelis–Menten‐type saturation kinetics. The vacuolar enriched‐membrane vesicles had a 46‐fold greater affinity and a 10‐fold greater transport activity with SGE than with SAG. We propose that in Arabidopsis, SAG is transported into the vacuole to serve as a long‐term storage form of SA while SGE, although also transported into the vacuole, is easily hydrolyzed to release the active hormone which can then be remobilized to other cellular locations.     

Tracking the dynamic interplay between bacterial and host factors during pathogen‐induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen‐induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram‐negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin‐3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high‐content and high‐throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.     

Trafficking of the Phosphoprotein PfCRT to the Digestive Vacuolar Membrane in Plasmodium falciparum

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site‐directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane.     

Vacuolar respiration of nitrate coupled to energy conservation in filamentous Beggiatoaceae

We show that the nitrate storing vacuole of the sulfide‐oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation.     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

On Food Vacuoles in Tetrahymena pyriformis GL

SYNOPSIS. The following problems concerning food vacuoles were studied by in vivo observations of Tetrahymena: (A) Formation of food vacuoles . The process may be divided into 4 stages. Stage 1—gradual growth of the limiting membrane of the open food vacuole (of short duration). Stage 2—"filling up" of the fully expanded vacuole (of long duration). Stage 3—"closing off" of the vacuole (of brief duration). Stage 4—initial movement of the detached vacuole away from the cy-tostome. The possible role of the oral components (apart from membranellar beating) in the process is discussed. (B) Change of pH in the food vacuole . After ingestion of heat-killed yeast stained with indicator dyes (neutral red, bromcresol purple, bromcresol green, bromphenol blue), the observed color changes indicate that pH is neutral in the forming vacuole as well as in newly formed vacuoles; that a pH value of 6.0–5.5 is reached after ∼ 5 min; and that the lowest pH value between 4.0 and 3.5 is reached after 1 hr. Before egestion the pH again increases. (C) Length of the digestive cycle . A determination of the time required to deplete the cells of labeled vacuoles formed during a short exposure, was attempted. Defecation was observed after 1/2 hr and it was frequent after 2 hr. About 25% and 50% of the labeled vacuoles were removed after 1 hr and 2 hr, respectively; however, labeled vacuoles may still be seen in some cells 6 hr after ingestion. The conclusion is that the digestive cycle lasts ∼ 2 hr and that egestion of undigestible material is a random process.     

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Summary Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.Abbreviations CLSM Confocal laser scanning microscopy - DIC differential interference contrast - LY Lucifer yellow CH - PM plasma membrane     

Differentiation of food vacuolar membranes during endocytosis in Tetrahymena

The origin and differentiation of Tetrahymena pyriformis food vacuolar membranes has been studied by freeze-fracture electron microscopy. By measuring the temperature needed to induce the onset of lipid phase separation (as inferred by the appearance of particle-free regions in replicas) and calculating the changes in average intramembrane particle distribution, a distinct modification of the vacuolar membrane could be observed from the time of its formation from disk-shaped vesicles to its maturation before egestion of its indigestible contents. Whereas the nascent vacuolar membrane first showed signs of phase separation at 9 degrees C, this temperature rose to 14 degrees C in the completed vacuole and then, after lysosomal fusion, eventually declined to 12 degrees C. The average membrane particle density on the PF face increased from 761 +/- 219 to 1,625 +/- 350 per micron 2 during membrane differentiation. Like other membranes of the cell, the vacuolar membrane underwent adaptive changes in its physical properties in cells maintained for several hours at low temperature. This exposure to low temperature caused an equal effect in vacuoles formed before, during, or after the temperature shift-down. Normal changes in the properties of the vacuolar membrane may have some bearing on its programmed sequence of fusion reactions.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

Assessment of the influence of confounding factors (weight,salinity) on the response of biomarkers in the estuarine polychaete Nereis diversicolor

The influences of salinity and body size on biochemical (activities of glutathione-S-transferase, lactate dehydrogenase (LDH), acetylcholinesterase and digestive enzymes amylase and CMCase), physiological (feeding and egestion rates, energy reserves) and behavioural (burrowing speed) biomarkers were examined in the infaunal polychaete Nereis diversicolor. Only a few biomarkers were affected, including increased egestion rate and activities of CMCase and LDH at higher salinity, and higher egestion rate in larger worms. These findings reinforce the status of N. diversicolor as a robust sentinel species for estuaries which are environments that are particularly productive but also particularly at risk.     

Inorganic phosphate uptake in intact vacuoles isolated from suspension-cultured cells of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don under varying Pi status

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H⁺ pumping, we examined the dependency of H⁺ pumping on plant Pi status. Both H⁺ pumping and the activities of the vacuolar H⁺-pumps, the V-type H⁺-ATPase and the H⁺-PPase were enhanced under low Pi status. Despite this increase in H⁺ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for the activation of Pi uptake into the vacuole under low Pi status are discussed. Miwa Ohnishi and Tetsuro Mimura contributed equally to this work.     

The Role of the Contractile Vacuole in the Osmoregulation of Tetrahymena pyriformis*

The relationship of cell size and contractile vacuole efflux to osmotic stress was studied in Tetrahymena pyriformis strain W, after transfer into fresh solutions iso- or hypoosmotic to the growth medium. Microscopic measurements of the cell and contractile vacuole dimensions, made with an image-sharing ocular at 27 C, allowed the calculation of the cell size and shape and the vacuolar efflux rate which provide a measure of osmoregulation. The contractile vacuole cycles have no homeostatic oscillations. In 0.03–0.10 osmolar solutions, the cell size and shape are constant while the vacuolar efflux rate has an inverse linear dependence upon extracellular osmolarity. Regression analyses indicate that for cells with systole faster than 0.1 sec (the major part of the population), it is only the final diastolic volume of the contractile vacuole that is related to osmotic stress while the frequency of systole is independent of osmotic stress and has a constant period of 7.7 ± 0.2 sec. Therefore, osmotic stress upon Tetrahymena is regulated by a corresponding change in the filling rate of its contractile vacuole to allow an unaltered cell size and shape. Kinetic measurements of vacuoles during diastole fit the model (dV/dt = K_1-K₂A), where (dV/dt) is the vacuolar filling rate and (A) is the vacuolar surface area. This dependence of vacuolar volume upon its surface area may be ascribed either to elastic components of the vacuolar membrane or to an increasing leakiness of this membrane during diastole. Mitochondrial inhibitors were used to observe the energy requirements of vacuolar operation and of intracellular secretion of water.     

Electrical properties ofValonia ventricosa

Summary The cytoplasmic electrical potential and membrane resistance of mature cells ofValonia ventricosa have been measured by inserting a microelectrode concentric with another electrode into the vacuole of the cell. The cytoplasmic region was investigated by advancing the microelectrode into the cell wall from the vacuolar side.The results revealed a unique region where the vacuolar electric potential and membrane resistance changed in a simultaneous single step to values close to zero. The measured potential always remained positive immediately after the step.At no time was a highly negative potential region encountered. Further penetration of the microelectrode revealed a low resistance negative potential region of –12.6±1.1 mV associated with the cell wall. Experiments were also carried out on aplanospores ofV. ventricosa to compare mature and immature cells. The chemical composition of the vacuolar and protoplasmic phases of mature cells was determined. The results agreed with previous results except that the Cl^– ion content of the protoplasm was significantly higher at 381±20 mmoles/liter (H₂O). It was concluded that mature cells ofValonia are significantly different from immature cells in that no highly negative potential cytoplasmic region was found in mature cells.It was considered that the measured step change in electric potential and membrane resistance occurred at the plasmalemma and that the tonoplast was a region of very low resistance. The implications of these findings in terms of models of ion transport intoValonia are discussed.     

The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 g/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C¹⁴ leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely under the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 g/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C¹⁴ leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed.     

Role of Vac8 in formation of the vacuolar sequestering membrane during micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.     

Role of Vac8 in Formation of the Vacuolar Sequestering Membrane during Micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.