Vacuole membrane fusion

Bayer, Martin; Reese, Christoph; Bühler, Susanne; Peters, Christopher M.; Mayer, Andreas

doi:10.1083/jcb.200212004

Cited by 168 publications

(74 citation statements)

References 96 publications

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“…These steps link Ca 2ϩ signaling to regulated neuronal membrane fusion, as elegantly reconstituted in a defined model reaction (51), but it remains unclear whether Ca 2ϩ is directly involved in regulating the fusion of other membranes, such as the vacuole, which lack synaptotagmin. One model of bilayer mixing during vacuole fusion posits a radially expanding, proteinaceous "fusion pore" of apposed, oligomerized vacuolar ATPase V0 sectors (52)(53)(54). Alternatively, there is evidence for a "hemifusion" transition state in membrane fusion consisting of a predominantly lipidic "neck" (55)(56)(57).…”

Section: Discussionmentioning

confidence: 99%

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

Starai¹,

Thorngren

Fratti³

et al. 2005

Journal of Biological Chemistry

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2؉ , ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab-and SNARE-dependent membrane fusion.

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Section: Discussionmentioning

confidence: 99%

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

Starai¹,

Thorngren

Fratti³

et al. 2005

Journal of Biological Chemistry

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“…2), are not well understood. Several factors have been suggested to participate in late stages of vacuole fusion, including protein phosphatase 1 (20), actin (23), subunits of the V 0 complex of the vacuolar proton ATPase (24), and the armadillo-repeat protein Vac8p (25,26). We find that protein and peptide ligands of phosphoinositides inhibit fusiondependent ALP maturation with postdocking kinetics, inhibit vacuole size increase, and do not inhibit ALP maturation in dilute detergent lysates (R. Fratti, A.J.M., N. Thorngren, and W.T.W., unpublished results).…”

Section: Discussionmentioning

confidence: 99%

Resolution of organelle docking and fusion kinetics in a cell-free assay

Merz

Wickner

2004

Proc. Natl. Acad. Sci. U.S.A.

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In vitro assays of compartment mixing have been key tools in the biochemical dissection of organelle docking and fusion. Many such assays measure compartment mixing through the enzymatic modification of reporter proteins. Homotypic fusion of yeast vacuoles is measured with a coupled assay of proteolytic maturation of pro-alkaline phosphatase (pro-ALP). A kinetic lag is observed between the end of docking, marked by the acquisition of resistance to anti-SNARE reagents, and ALP maturation. We therefore asked whether the time taken for pro-ALP maturation adds a kinetic lag to the measured fusion signal. Prb1p promotes ALP maturation; overproduction of Prb1p accelerates ALP activation in detergent lysates but does not alter the measured kinetics of docking or fusion. Thus, the lag between docking and ALP activation reflects a lag between docking and fusion. Many vacuoles in the population undergo multiple rounds of fusion; methods are presented for distinguishing the first round of fusion from ongoing rounds of fusion. A simple kinetic model distinguishes between two rates, the rate of fusion and the rate at which fusion competence is lost, and allows estimation of the number of rounds of fusion completed.Pho8p ͉ protease B ͉ SNARE ͉ Rab͞Ypt ͉ tethering M embrane docking and fusion events have been measured by many methods. Among the most commonly used are in vitro, coupled enzymatic assays of compartment mixing between two vesicle populations (1). One vesicle population contains a reporter substrate, and the other vesicle population contains an enzyme that can covalently modify the reporter substrate. We call vesicles containing the modifying enzyme ''effector'' vesicles and vesicles containing the substrate ''reporter'' vesicles. Fusion causes content mixing between effector and reporter vesicles, allowing the enzyme access to the reporter substrate. The amount of reporter modified thereby indicates the extent of fusion.Coupled transport and fusion assays have made possible the identification of molecules that mediate vesicle budding, docking and fusion, and the dissection of the mechanisms by which these molecules act. However, coupled assays have potential limitations. Treatments that inhibit the reporter system may be interpreted incorrectly as fusion inhibitors. Moreover, if the enzymatic processing of the reporter substrate is slow relative to the docking or fusion reaction of interest, the kinetics of the processing event will dominate, and thus obscure, the kinetics of fusion.We study yeast vacuole͞vacuole homotypic fusion. A key tool in our work is a cell-free, coupled enzymatic assay of vacuole fusion (2). In this assay, the reporter substrate is the enzyme alkaline phosphatase (ALP), encoded by the PHO8 gene. ALP is synthesized as an inactive precursor, pro-ALP, and is processed into an active form by proteolytic cleavage (3, 4). The reporter vacuoles contain pro-ALP but are protease deficient. The effector vacuoles are protease replete but lack pro-ALP. Content mixing between effector and reporter vacuo...

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“…PHO81 encodes a cyclin-dependent kinase that initiates the PHO pathway (60), and PHO84 encodes a high affinity inorganic phosphate transporter (61). VTC1, VTC3, and VTC4 gene products are involved in polyphosphate accumulation by the vacuole (59) but also function together in a complex during late stages of vacuole fusion (62)(63)(64). It is interesting that two additional Criteria for classifying Ino2p-Ino4p and UPR target genes are described under "Results."…”

Section: Analysis Of Genome-wide Expression Regulated By Inositolmentioning

confidence: 99%

Genome-wide Analysis Reveals Inositol, Not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast

Jesch

Zhao

Wells

et al. 2005

Journal of Biological Chemistry

120

194

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In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was used to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination of inositol and choline increased the number of repressed genes compared with inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild-type cells. One set of genes contained the UAS INO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another nonoverlapping set of genes was coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but was not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, whereas choline plays a minor role.Phospholipids are the key structural elements of membranebounded organelles and play important roles in signaling and membrane trafficking pathways. Each membrane compartment is composed of a unique set of phospholipids whose biophysical properties contribute to the function of each organelle. Phospholipid metabolism is highly regulated by the cell, ensuring the biogenesis and growth of membranes by coordinating the relative rates of synthesis of individual phospholipids with numerous factors, such as the availability of exogenous supplies of phospholipid precursors, growth stage, and membrane trafficking (1, 2).

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Vacuole membrane fusion

Cited by 168 publications

References 96 publications

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae

Resolution of organelle docking and fusion kinetics in a cell-free assay

Genome-wide Analysis Reveals Inositol, Not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast

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