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.     

Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p.     

A truncated form of the Pho80 cyclin of Saccharomyces cerevisiae induces expression of a small cytosolic factor which inhibits vacuole inheritance.

Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of a soluble inhibitory factor. When mixed with wild-type cytosol, vac5-1 cytosol inhibits the activity of wild-type cytosol. vac5-1 cytosol treated with trypsin or papain is still able to inhibit the activity of Aid-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein traction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-l small-molecule fraction retains the full ability to inhibit fusion. Thus, the vac5-1 allele of PHO80 induces the synthesis of a small molecule that is an inhibitor of vacuole inheritance.     

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a segregation structure) into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one low molecular weight activity (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.     

A Heterodimer of Thioredoxin and IB2 Cooperates with Sec18p (NSF) to Promote Yeast Vacuole Inheritance

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as I^B₂, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.     

Docking of Yeast Vacuoles Is Catalyzed by the Ras-like GTPase Ypt7p after Symmetric Priming by Sec18p (NSF)

Vacuole inheritance in yeast involves the formation of tubular and vesicular “segregation structures” which migrate into the bud and fuse there to establish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and microscopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacuoles must be primed for docking by the action of Sec17p (α-SNAP) and Sec18p (NSF). Priming is necessary for both fusion partners. It produces a labile state which requires rapid docking in order to lead productively to fusion. In addition to Sec17p/Sec18p, docking requires the activity of the Ras-like GTPase Ypt7p. Unlike Sec17p/Sec18p, which must act before docking, Ypt7p is directly involved in the docking process itself.     

Proteins needed for vesicle budding from the Golgi complex are also required for the docking step of homotypic vacuole fusion

Vam2p/Vps41p is known to be required for transport vesicles with vacuolar cargo to bud from the Golgi. Like other VAM-encoded proteins, which are needed for homotypic vacuole fusion, we now report that Vam2p and its associated protein Vam6p/Vps39p are needed on each vacuole partner for homotypic fusion. In vitro vacuole fusion occurs in successive steps of priming, docking, and membrane fusion. While priming does not require Vam2p or Vam6p, the functions of these two proteins cannot be fulfilled until priming has occurred, and each is required for the docking reaction which culminates in trans-SNARE pairing. Consistent with their dual function in Golgi vesicle budding and homotypic fusion of vacuoles, approximately half of the Vam2p and Vam6p of the cell are recovered from cell lysates with purified vacuoles.     

Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro

Vacuole inheritance in Saccharomyces cerevisiae can be reconstituted in vitro using isolated organelles, cytosol, and ATP. Using the requirements of the reaction and its susceptibility to inhibitors, we have divided the in vitro reaction into four biochemically distinct, sequential subreactions. Stage I requires exposure of vacuoles to solutions of moderate ionic strength. Stage II requires "stage I" vacuoles and cytosol. In stage III, stage II vacuoles react with ATP. Finally, during stage IV, stage III vacuoles at a certain, minimal concentration complete the fusion reaction without further requirement for any soluble components. Reagents that inhibit the overall vacuole inheritance reaction block distinct stages. Stage III of the reaction is sensitive to the proton ionophore CCCP, to inhibitors of the vacuolar ATPase such as bafilomycin A1, and to the ATP-hydrolyzing enzyme apyrase, suggesting that an electrochemical potential across the vacuolar membrane is required during this stage. Inhibition studies with the amphiphilic peptide mastoparan and GTP gamma S suggest that GTP-hydrolyzing proteins might also be involved during this stage. Microcystin-LR, a specific inhibitor of protein phosphatases of type 1 and 2A, inhibits stage IV of the inheritance reaction, indicating that a protein dephosphorylation event is necessary for fusion. The definition of these four stages may allow the development of specific assays for the factors which catalyze each of the consecutive steps of the in vitro reaction.     

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.     

Phagosome fusion vesicles of paramecium. I. Thin-section morphology

Ultrastructural studies of the digestive system of Paramecium caudatum focusing on the first 5 min of digestive-vacuole age reveal a set of vesicles, named phagosome fusion vesicles (PFVs), which fuse with the digestive vacuole just after the vacuoles are released from the cytopharynx and concomitant with vacuole acidification. Serial thin-sections of vacuoles labeled with horseradish peroxidase (HRP) and/or latex beads in pulse-chase studies were observed. PFVs, irregularly shaped, electron-translucent vesicles ranging from a small diameter to over 1 micro, are first seen in the region of the cytopharynx where they bind to the nascent vacuole membrane. Within 30 sec of vacuole release the PFVs fuse with the vacuole where they remain for a brief time connected to the vacuole by a narrow annulus. HRP-reaction product is found in vacuoles but not in PFVs before PFVs fuse with the vacuoles. After fusion with PFVs HRP is quickly inactivated. Tubular extensions of vacuole membrane then form between the fused PFVs. By 3 to 5 min both PFVs and tubules disappear from the vacuole surface and lysosomes appear in their place. We believe the tubules are pinched off as PFV membrane is being added to the vacuole. Microfilaments coat the membrane during all these dynamic events. Since the pH of the vacuole becomes acid during the first few minutes, we are now looking for a direct correlation between PFV fusion and acidification.     

Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.     

Vacuole inheritance regulates cell size and branching frequency of Candida albicans hyphae

Hyphal growth of Candida albicans is characterized by asymmetric cell divisions in which the subapical mother cell inherits most of the vacuolar space and becomes cell cycle arrested in G1, while the apical daughter cell acquires most of the cell cytoplasm and progresses through G1 into the next mitotic cell cycle. Consequently, branch formation in hyphal compartments is delayed until sufficient cytoplasm is synthesized to execute the G1 'START' function. To test the hypothesis that this mode of vacuole inheritance determines cell cycle progression and therefore the branching of hyphae, eight tetracycline-regulated conditional mutants were constructed that were affected at different stages of the vacuole inheritance pathway. Under repressing conditions, vac7 , vac8 and fab1 mutants generated mycelial compartments with more symmetrically distributed vacuoles and increased branching frequencies. Repression of VAC1 , VAM2 and VAM3 resulted in sparsely branched hyphae, with large vacuoles and enlarged hyphal compartments. Therefore, during hyphal growth of C. albicans the cell cycle, growth and branch formation can be uncoupled, resulting in the investment of cytoplasm to support hyphal extension at the expense of hyphal branching.     

Architectural remodeling of the tonoplast during fluid-phase endocytosis

During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.     

ATP is released from autophagic vesicles to the extracellular space in a VAMP7-dependent manner

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called autophagosome. SNAREs proteins are key molecules of the vesicle fusion machinery. Our results indicate that in a mammalian tumor cell line a subset of VAMP7 (V-SNARE)-positive vacuoles colocalize with LC3 at the cell periphery (focal adhesions) upon starvation. The re-distribution of VAMP7 positive structures is a microtubule-dependent event, with the participation of the motor protein KIF5 and the RAB7 effector RILP. Interestingly, most of the VAMP7-labeled vesicles were loaded with ATP. Moreover, in cells subjected to starvation, these structures fuse with the plasma membrane to release the nucleotide to the extracellular medium. Summarizing, our results show the molecular components involved in the release of ATP to extracellular space, which is recognized as an important autocrine/paracrine signal molecule that participates in the regulation of several cellular functions such as immunogenicity of cancer cell death or inflammation     

ATP is released from autophagic vesicles to the extracellular space in a VAMP7-dependent manner

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called autophagosome. SNAREs proteins are key molecules of the vesicle fusion machinery. Our results indicate that in a mammalian tumor cell line a subset of VAMP7 (V-SNARE)-positive vacuoles colocalize with LC3 at the cell periphery (focal adhesions) upon starvation. The re-distribution of VAMP7 positive structures is a microtubule-dependent event, with the participation of the motor protein KIF5 and the RAB7 effector RILP. Interestingly, most of the VAMP7-labeled vesicles were loaded with ATP. Moreover, in cells subjected to starvation, these structures fuse with the plasma membrane to release the nucleotide to the extracellular medium. Summarizing, our results show the molecular components involved in the release of ATP to extracellular space, which is recognized as an important autocrine/paracrine signal molecule that participates in the regulation of several cellular functions such as immunogenicity of cancer cell death or inflammation     

薏苡胚乳细胞化的超微结构观察

采用透射电镜对薏苡早期的胚乳细胞化进行了研究,在胚乳游离核时期,胚乳游离核及细胞质绕中央细胞分布,游离核间没有发现胚囊壁内突、成膜体等结构。胚乳细胞化过程中初始垂周壁形成过程如下：（１）胚乳细胞质中出现液泡,使细胞质和核向中央液泡推进：（２）一对相邻细胞核间液泡成对存在,且呈垂周分布,而且两液泡间的细胞质很狭窄;（３）在这狭窄的细胞质中出现成行排列的小泡;（４）小泡融合形成细胞板,细胞板悬于两液泡     

Concanavalin A and the in vitro induction in macrophages of vacuolation and lysosomal enzyme synthesis

Concanavalin A (ConA) induced extensive vacuolation in mouse peritoneal macrophages. Electron microscopic observations on thin sections reveal that the vacuoles are essentially empty except for minute vesicles attached to their inner periphery. The vacuoles consist of irregular structures and are heterogeneous in size distribution. ConA-induced vacuoles exhibit high acid phosphatase activity, suggesting fusion between vacuoles and lysosomes. Induction of acid phosphatase in ConA-treated macrophages was studied under several cultivation conditions. ConA-treated macrophage cultures responded in increase in acid phosphatase activity early after exposure to the lectin, a significant increase recorded already after 1 h. When cultivated in 1% serum medium for 48 h, ConA-treated macrophages exhibit twice the activity of acid phosphatase at zero time as well as that of non-treated control cultures. The effect of ConA on thioglycolate-stimulated mouse peritoneal macrophages was also studied. Vacuole formation resulting from lectin binding and internalization is discussed in terms of possible lectin effects on membrane fluidity, fusion capacity, surface to volume conservation during vacuole formation, fusion of vacuoles with lysosomes and intravacuolar lysosomal enzyme activities. The phenomenon of lysosomal enzyme induction as a result of ConA treatment is being correlated with enzyme induction due to other stimuli.     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion

In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7 and SNARE-mediated pairing of vacuoles; and fusion, mediated by calmodulin/V0/t-SNARE interactions. Defects in catalysts of each stage result in fragmented (unfused) vacuoles. Strains with deletions in any of ERG genes 3-6, lacking normal ergosterol biosynthesis, have fragmented vacuoles. The ergosterol ligands filipin, nystatin and amphotericin B block the in vitro fusion of vacuoles from wild-type cells. Each of these inhibitors acts at the priming stage to inhibit Sec17p release from vacuoles. A reversible delay in Sec18p action prevents vacuoles from acquiring resistance to any of these three drugs, confirming that their action is on the normal fusion pathway. Ergosterol or cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and the in vitro fusion of ergDelta vacuoles requires added sterol. The need for ergosterol for vacuole priming underscores the role of lipids in organizing the membrane elements of this complex reaction.