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At least 19 recordsLinked to original sources

PubMed2001

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles.

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.

Base Sequence↗

PubMed2000

Transport of proteins to the yeast vacuole: autophagy, cytoplasm-to-vacuole targeting, and role of the vacuole in degradation.

The vacuole/lysosome performs a central role in degradation. Proteins and organelles are transported to the vacuole by selective and non-selective pathways. Transport to the vacuole by autophagy is the primary mode for degradation of cytoplasmic constituents under starvation conditions. Autophagy overlaps mechanistically and genetically with a biosynthetic pathway termed Cvt (Cytoplasm-to-vacuole targeting) that operates under vegetative conditions to transport the resident vacuolar hydrolase aminopeptidase I (API). API import has been dissected to reveal the action of a novel mechanism that transports cargo within double-membrane vesicles. Recent work has uncovered molecular components involved in autophagy and the Cvt pathway.

Aminopeptidases↗

PubMed1997

Vacuole segregation in the Saccharomyces cerevisiae vac2-1 mutant: structural and biochemical quantification of the segregation defect and formation of new vacuoles.

The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10, 1741-1748] shifted to non-permissive temperature (37 degrees C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37 degrees C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10-15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37 degrees C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding.

Carboxypeptidases↗

PubMed1998

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.

In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.

Animals↗

PubMed2006

Enolase activates homotypic vacuole fusion and protein transport to the vacuole in yeast.

Membrane fusion and protein trafficking to the vacuole are complex processes involving many proteins and lipids. Cytosol from Saccharomyces cerevisiae contains a high Mr activity, which stimulates the in vitro homotypic fusion of isolated yeast vacuoles. Here we purify this activity and identify it as enolase (Eno1p and Eno2p). Enolase is a cytosolic glycolytic enzyme, but a small portion of enolase is bound to vacuoles. Recombinant Eno1p or Eno2p stimulates in vitro vacuole fusion, as does a catalytically inactive mutant enolase, suggesting a role for enolase in fusion that is separate from its glycolytic function. Either deletion of the non-essential ENO1 gene or diminished expression of the essential ENO2 gene causes vacuole fragmentation in vivo, reflecting reduced fusion. Combining an ENO1 deletion with ENO2-deficient expression causes a more severe fragmentation phenotype. Vacuoles from enolase 1 and 2-deficient cells are unable to fuse in vitro. Immunoblots of vacuoles from wild type and mutant strains reveal that enolase deficiency also prevents normal protein sorting to the vacuole, exacerbating the fusion defect. Band 3 has been shown to bind glycolytic enzymes to membranes of mammalian erythrocytes. Bor1p, the yeast band 3 homolog, localizes to the vacuole. Its loss results in the mislocalization of enolase and other vacuole fusion proteins. These studies show that enolase stimulates vacuole fusion and that enolase and Bor1p regulate selective protein trafficking to the vacuole.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2004

Geminating pollen has tubular vacuoles, displays highly dynamic vacuole biogenesis, and requires VACUOLESS1 for proper function.

Vacuoles perform multiple functions in plants, and VCL1 (VACUOLESS1) is essential for biogenesis with loss of expression in the vcl1 mutant leading to lethality. Vacuole biogenesis plays a prominent role in gametophytes, yet is poorly understood. Given the importance of VCL1, we asked if it contributes to vacuole biogenesis during pollen germination. To address this question, it was essential to first understand the dynamics of vacuoles. A tonoplast marker, delta-TIP::GFP, under a pollen-specific promoter permitted the examination of vacuole morphology in germinating pollen of Arabidopsis. Our results demonstrate that germination involves a complex, yet definable, progression of vacuole biogenesis. Pollen vacuoles are extremely dynamic with remarkable features such as elongated (tubular) vacuoles and highly mobile cytoplasmic invaginations. Surprisingly, vcl1 did not adversely impact vacuole morphology in pollen germinated in vitro. To focus further on VCL1 in pollen, reciprocal backcrosses demonstrated reduced transmission of vcl1 through male gametophytes, indicating that vcl1 was expressive after germination. Interestingly, vcl1 affected the fertility of female gametophytes that undergo similarly complex vacuole biogenesis. Our results indicate that vcl1 is lethal in the sporophyte but is not fully expressive in the gametophytes. They also point to the complexity of pollen vacuoles and suggest that the mechanism of vacuole biogenesis in pollen may differ from that in other plant tissues.

Arabidopsis↗

PubMed1998

Vac8p, a vacuolar protein with armadillo repeats, functions in both vacuole inheritance and protein targeting from the cytoplasm to vacuole.

During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells. This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p. A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration. Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance. Both myristoylation and palmitoylation are required for complete Vac8p localization. Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential. Thus, palmitoylation appears to play a more direct role in vacuole inheritance. Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats. Arm repeats are thought to mediate protein-protein interactions, and many Arm proteins have multiple functions. This is also true for Vac8p. In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole. Mutant analysis demonstrates that Vac8p functions separately in these two processes. Vac8p cosediments with actin filaments. Vac8p is related to beta-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton. In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.

Actins↗

PubMed2003

Cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of Helicobacter pylori vacuolating cytotoxin activity that are each dependent on membrane channel formation.

Helicobacter pylori vacuolating toxin (VacA) is a secreted toxin that is reported to produce multiple effects on mammalian cells. In this study, we explored the relationship between VacA-induced cellular vacuolation and VacA-induced cytochrome c release from mitochondria. Within intoxicated cells, vacuolation precedes cytochrome c release and occurs at lower VacA concentrations, indicating that cellular vacuolation is not a downstream consequence of cytochrome c release. Conversely, bafilomycin A1 blocks VacA-induced vacuolation but not VacA-induced cytochrome c release, which indicates that cytochrome c release is not a downstream consequence of cellular vacuolation. Acid activation of purified VacA is required for entry of VacA into cells, and correspondingly, acid activation of the toxin is required for both vacuolation and cytochrome c release, which suggests that VacA must enter cells to produce these two effects. Single amino acid substitutions (P9A and G14A) that ablate vacuolating activity and membrane channel-forming activity render VacA unable to induce cytochrome c release. Channel blockers known to inhibit cellular vacuolation and VacA membrane channel activity also inhibit cytochrome c release. These data indicate that cellular vacuolation and mitochondrial cytochrome c release are two independent outcomes of VacA intoxication and that both effects are dependent on the formation of anion-selective membrane channels.

Anions↗

PubMed1996

Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly.

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.

Aminopeptidases↗

PubMed1995

The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance.

In the budding yeast Saccharomyces cerevisiae, vacuoles are inherited by the projection of vesicles and tubules from the mother-cell vacuole into the growing daughter cell during the S phase. These vesicles then fuse and form the daughter-cell organelle. We have described previously in vitro reactions of the formation of vacuole-derived segregation structures and of vacuole-vacuole fusion. Homotypic vacuole fusion requires cytosol, ATP and a physiological temperature, and is sensitive to GTPase inhibitors. These reactions are divisible into early stages which require ATP and cytosol, and late stages which require neither. Here, we report that Ypt7p, a ras-like GTPase implicated previously in endocytosis in yeast, is largely localized to the vacuole and is required on both partners during the in vitro vacuole fusion reaction. The in vitro fusion reaction is inhibited either by Gdi1p, which extracts the GDP-bound form of ras-like GTPases from membranes, or by antibodies specific for Ypt7p. The presence of anti-Ypt7p during the early stages of the reaction inhibits the development of cytosol- and ATP-independent intermediates. Although cytosol and ATP are no longer needed for the late stage of vacuole inheritance in vitro, the inhibition of this late stage by anti-Ypt7p or Gdi1p requires the continued presence of ATP and cytosol. Ypt7p is the first GTPase for which a direct role in organelle inheritance has been established.

Base Sequence↗

PubMed1991

Diffusion resistances between ADH-induced vacuoles and the extracellular space in rabbit collecting duct: evidence that most vacuoles are intracellular, endocytic compartments.

Large vacuoles form in the renal collecting duct following the onset of antidiuretic hormone (ADH)-stimulated water reabsorption. The aim of the present study was to test two alternative hypotheses regarding the origins of these structures: (1) the vacuoles constitute basilar, extracellular spaces that dilate as water flows through these spaces from cells into the peritubular compartment; or (2) the vacuoles represent intracellular, endocytic compartments that dilate during water reabsorption due to enhanced fluid phase endocytosis. Fluorescence-digital imaging microscopy was used to visualize the uptake into vacuoles of a hydrophilic fluorochrome (6 methoxy-N-[3 sulfopropyl] quinolinium) whose fluorescence is markedly quenched by halides. During their formation, most vacuoles (67%) accumulated the fluorochrome from the peritubular bath and trapped the dye well after (greater than 60 min) washing it from the bath. The spatial pattern of fluorescence within individual vacuoles indicated that the dye was trapped within these structures as a fluid-phase marker and was not bound to the vacuole margins. The fluorescence of dye trapped within vacuoles was virtually unaltered by changes in peritubular Cl- or Br- concentration that elicit dramatic quenching of dye-fluorescence in bulk solution, as expected if there exists a high diffusion resistance between the interiors of these structures and the peritubular space. These results indicate that most ADH-induced vacuoles represent endocytic compartments that are not directly connected to the extracellular space.

Animals↗

PubMed2002

Plasma membrane cholesterol modulates cellular vacuolation induced by the Helicobacter pylori vacuolating cytotoxin.

The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using beta-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, beta-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.

Animals↗

PubMed1997

Helicobacter pylori vacuolating toxin accumulates within the endosomal-vacuolar compartment of cultured gastric cells and potentiates the vacuolating activity of ammonia.

This study explored the relationship between vacuolating toxin and ammonia in the genesis of Helicobacter pylori-induced vacuolation in cultured human gastric cells and investigated the intracellular sites of toxin accumulation. Neutral red dye uptake and electron microscopy were used in the investigation of the respective roles of, and of the reciprocal interaction between, toxin and ammonia in cell vacuolation and ultrastructural immunocytochemistry was used for the identification of the intracellular sites of internalized toxin. Toxin was found to cause an expansion of the endosomal compartment, where it accumulates after cellular internalization. However, toxin does not form large, neutral red-positive vacuoles unless combined with ammonia, whose moderate vacuolating activity is markedly potentiated by the toxin. It is concluded that the toxin accumulated within the endosomal compartment alters the morphology and function of this organelle and plays a permissive role towards cell vacuolation, possibly by increasing the accumulation of protonated ammonia within endosomes. In turn, ammonia induces excessive dilatation of the endosomes with reciprocal fusion of their membranes, thus causing cytoplasmic vacuolation.

Ammonia↗

PubMed2003

Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase.

In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.

Enzyme Activation↗

PubMed1968

Autophagic vacuoles produced in vitro. II. Studies on the mechanism of formation of autophagic vacuoles produced by chloroquine.

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1-2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.

Animals↗

PubMed1991

vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting.

We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.

Aspartic Acid Endopeptidases↗

PubMed2004

Brefeldin A enhances Helicobacter pylori vacuolating cytotoxin-induced vacuolation of epithelial cells.

Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region. We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA. The effect is BFA dose-dependent and occurs within 2.5 h. These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently. This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo.

Animals↗

PubMed1999

Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin.

Helicobacter pylori secretes a cytotoxin (VacA) that induces the formation of large vacuoles originating from late endocytic vesicles in sensitive mammalian cells. Although evidence is accumulating that VacA is an A-B toxin, distinct A and B fragments have not been identified. To localize the putative catalytic A-fragment, we transfected HeLa cells with plasmids encoding truncated forms of VacA fused to green fluorescence protein. By analyzing truncated VacA fragments for intracellular vacuolating activity, we reduced the minimal functional domain to the amino-terminal 422 residues of VacA, which is less than one-half of the full-length protein (953 amino acids). VacA is frequently isolated as a proteolytically nicked protein of two fragments that remain noncovalently associated and retain vacuolating activity. Neither the amino-terminal 311 residue fragment (p33) nor the carboxyl-terminal 642 residue fragment (p70) of proteolytically nicked VacA are able to induce cellular vacuolation by themselves. However, co-transfection of HeLa cells with separate plasmids expressing both p33 and p70 resulted in vacuolated cells. Further analysis revealed that a minimal fragment comprising just residues 312-478 functionally complemented p33. Collectively, our results suggest a novel molecular architecture for VacA, with cytosolic localization of both fragments of nicked toxin required to mediate intracellular vacuolating activity.

Anti-Bacterial Agents↗