scholarly journals Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion

2020 ◽  
Vol 21 (12) ◽  
pp. 4530 ◽  
Author(s):  
Carmen Siebenaller ◽  
Benedikt Junglas ◽  
Annika Lehmann ◽  
Nadja Hellmann ◽  
Dirk Schneider
Keyword(s):  
Membrane Fusion ◽  
Thylakoid Membrane ◽  
Physiological Function ◽  
Membrane Lipids ◽  
Membrane System ◽  
Dual Function ◽  
Charged Membrane ◽  
Proton Leakage ◽  
Chloroplast Stroma

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

scholarly journals A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein.

1991 ◽  
Vol 112 (4) ◽  
pp. 603-613 ◽  
Author(s):  
L A Payan ◽  
K Cline
Keyword(s):  
Membrane Protein ◽  
Thylakoid Membrane ◽  
Gel Filtration ◽  
Chloroplast Envelope ◽  
Membrane Insertion ◽  
Protein Factor ◽  
Stromal Component ◽  
Thylakoid Protein ◽  
Chloroplast Stroma

The light-harvesting chlorophyll a/b protein (LHCP) is an approximately 25,000-D thylakoid membrane protein. LHCP is synthesized in the cytosol as a precursor and must translocate across the chloroplast envelope before becoming integrally associated with the thylakoid bilayer. Previous studies demonstrated that imported LHCP traverses the chloroplast stroma as a soluble intermediate before thylakoid insertion. Here, examination of this intermediate revealed that it is a stable, discrete approximately 120,000-D species and thus either an LHCP oligomer or a complex with another component. In vitro-synthesized LHCP can be converted to a similar form by incubation with a stromal extract. The stromal component responsible for this conversion is proteinaceous as evidenced by its inactivation by heat, protease, and NEM. Furthermore, the conversion activity coelutes from a gel filtration column with a stromal protein factor(s) previously shown to be necessary for LHCP integration into isolated thylakoids. Conversion of LHCP to the 120-kD form prevents aggregation and maintains its competence for thylakoid insertion. However, conversion to this form is apparently not sufficient for membrane insertion because the isolated 120-kD LHCP still requires stroma to complete the integration process. This suggests a need for at least one more stroma-mediated reaction. Our results explain how a hydrophobic thylakoid protein remains soluble as it traverses the aqueous stroma. Moreover, they describe in part the function of the stromal requirement for insertion into the thylakoid membrane.

How calcium may cause exocytosis in sea urchin eggs

1987 ◽  
Vol 7 (5) ◽  
pp. 383-397 ◽  
Author(s):  
Michael Whitaker
Keyword(s):  
Membrane Fusion ◽  
Lipid Bilayers ◽  
Secretory Granule ◽  
Sea Urchin ◽  
Surface Potential ◽  
Membrane Lipids ◽  
Electrical Potential ◽  
Sea Urchin Eggs ◽  
Granule Membrane

The process of secretory granule-plasma membrane fusion can be studied in sea urchin eggs. Micromolar calcium concentrations are all that is required to bring about exocytosis in vitro. I discuss recent experiments with sea urchin eggs that concentrate on the biophysical aspects of granule-membrane fusion. The backbone of biological membranes is the lipid bilayer. Sea urchin egg membrane lipids have negatively charged head groups that give rise to an electrical potential at the bilayer-water interface. We have found that this surface potential can affect the calcium required for exocytosis. Effects on the surface potential may also explain why drugs like trifluoperazine and tetracaine inhibit exocytosis: they absorb to the bilayer and reduce the surface potential. The membrane lipids may also be crucial to the formation of the exocytotic pore through which the secretory granule contents are released. We have measured calcium-induced production of the lipid, diacylglycerol. This lipid can induce a phase transition that will promote fusion of apposed lipid bilayers. The process of exocytosis involves the secretory granule core as well as the lipids of the membrane. The osmotic properties of the granule contents lead to swelling of the granule during exocytosis. Swelling promotes the dispersal of the contents as they are extruded through the exocytotic pore. The movements of water and ions during exocytosis may also stabilize the transient fusion intermediate and consolidate the exocytotic pore as fusion occurs.

scholarly journals H2O2 Induces Association of RCA with the Thylakoid Membrane to Enhance Resistance of Oryza meyeriana to Xanthomonas oryzae pv. oryzae

Plants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 351 ◽  
Author(s):  
Mei ◽  
Yang ◽  
Ye ◽  
Liang ◽  
Wang ◽  
...  
Keyword(s):  
Thylakoid Membrane ◽  
Resistance Mechanism ◽  
Rubisco Activase ◽  
Universal Function ◽  
Xanthomonas Oryzae ◽  
Cultivated Rice ◽  
Bisphosphate Carboxylase ◽  
Chloroplast Stroma ◽  
Oryza Meyeriana

Oryza meyeriana is a wild species of rice with high resistance to Xanthomonas oryzae pv. oryzae (Xoo), but the detailed resistance mechanism is unclear. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is an important enzyme that regulates photosynthesis by activating Rubisco. We have previously reported that Xoo infection induced the relocation of RCA from the chloroplast stroma to the thylakoid membrane in O. meyeriana, but the underlying regulating mechanism and physiological significance of this association remains unknown. In this study, “H2O2 burst” with rapid and large increase in the amount of H2O2 was found to be induced by Xoo invasion in the leaves of O. meyeriana. 3, 3-diaminobenzidine (DAB) and oxidative 2, 7-Dichlorodi-hydrofluorescein diacetate (H2DCFDA) staining experiments both showed that H2O2 was generated in the chloroplast of O. meyeriana, and that this H2O2 generation as well as Xoo resistance of the wild rice were dramatically dependent on light. H2O2, methyl viologen with light, and the xanthine-xanthine oxidase system all induced RCA to associate with the thylakoid membrane in vitro, which showed that H2O2 could induce the relocation of RCA. In vitro experiments also showed that H2O2 induced changes in both the RCA and thylakoid membrane that were required for them to associate and that this association only occurred in O. meyeriana and not in the susceptible cultivated rice. These results suggest that the association of RCA with the thylakoid membrane helps to protect the thylakoid membrane against oxidative damage from H2O2. Therefore, in addition to its universal function of activating Rubisco, RCA appears to play a novel role in the resistance of O. meyeriana to Xoo.

scholarly journals The role of PI(4,5)P2 and synaptotagmin in membrane fusion - an in vitro study

2021 ◽  
Author(s):  
Joern Dietz ◽  
Marieelen Oelkers ◽  
Raphael Hubrich ◽  
Angel Perez-Lara ◽  
Reinhard Jahn ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Lipids ◽  
In Vitro Study ◽  
Snare Proteins ◽  
Large Unilamellar Vesicles ◽  
Intermediate States ◽  
Synaptotagmin 1 ◽  
Fusion Efficiency

Synaptotagmin-1 (syt-1) is known to trigger fusion of neuronal synaptic vesicles with the presynaptic membrane by recognizing acidic membrane lipids. In particular, binding to PI(4,5)P2 is believed to be crucial for its function as a calcium sensor. We propose a mechanism for syt-1 to interact with anionic bilayers and promote fusion in the presence of SNARE proteins. We found that in the absence of Ca2+ the binding of syt-1 to membranes depends on the PI(4,5)P2 content. Addition of Ca2+ switches the interaction forces from weak to strong eventually exceeding the cohesion of the C2A domain, while the interaction between PI(4,5)P2 and the C2B domain was preserved even in the absence of Ca2+ or phosphatidylserine. Fusion of large unilamellar vesicles equipped with syt-1 and synaptobrevin with freestanding target membranes composed of PS/PI(4,5)P2 show an increased fusion speed, and by effective suppression of stalled intermediate states, a larger number of full fusion events. Fusion efficiency could be maximized when irreversible docking is additionally prevented by addition of multivalent anions. The picture that emerges is that syt-1 remodels the membrane in the presence of calcium and PIP2, thereby substantially increasing the efficiency of membrane fusion by avoiding stalled intermediate states.

scholarly journals Proteins Needed for Vesicle Budding from the Golgi Complex Are Also Required for the Docking Step of Homotypic Vacuole Fusion

2000 ◽  
Vol 148 (6) ◽  
pp. 1223-1230 ◽  
Author(s):  
Albert Price ◽  
William Wickner ◽  
Christian Ungermann
Keyword(s):  
Membrane Fusion ◽  
Golgi Complex ◽  
Golgi Vesicle ◽  
Dual Function ◽  
Vesicle Budding ◽  
Cell Lysates ◽  
Vacuole Fusion ◽  
Transport Vesicles ◽  
In Trans

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.

scholarly journals Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: trigger analyses

1980 ◽  
Vol 46 (1) ◽  
pp. 41-60
Author(s):  
H. Matt ◽  
H. Plattner ◽  
K. Reichel ◽  
M. Lefort-Tran ◽  
J. Beisson
Keyword(s):  
Cell Membrane ◽  
Atpase Activity ◽  
Membrane Fusion ◽  
Dual Function ◽  
Genetic Dissection ◽  
Paramecium Tetraurelia ◽  
Attachment Sites ◽  
Ca2 Channels

A variety of trigger procedures were applied to analyse the exocytotic capability of different Paramecium tetraurelia strains. 7,S K 40I, kin 24I, and 9 (18 degrees C) are capable of exocytosis (permissive strains), in contrast to nd 6, nd 7, nd 9 (27 degrees C), tam 38 and ftb A, although all procedures used enhance [Ca2+]i in the cytoplasm of all strains tested and although strains nd 6, nd 7 and nd 9 (27 degrees C) contain a full set of morphologically normal trichocysts attended to the cell membrane. The results show that only those strains are permissive which were shown previously to contain a rosette of membrane-integrated particles and a Ca2+-ATPase activity in the cell membrane over the trichocyst attachment (exocytosis) sites. The results from trigger experiments with permissive and non-permissive strains would be compatible with a dual function of rosette particles as Ca2+ pumps and Ca2+ channels. Nevertheless, the latter aspect remains uncertain since we show that experiments along these lines published by others (introducing a Ca2+ ionophore from the outside) involve a solvent-induced artifact (pseudoexocytosis: matrix stretching in the absence of membrane fusion). In all strains, except for tam 38 and ftb A (which have abnormal trichocysts incapable of being attached to the cell membrane), the isolated trichocyst matrix can be transferred from the contracted to the expanded state in vitro with certain trigger procedures. Our data clearly show that an increase of [Ca2+]i in the cytoplasm is not sufficient for exocytosis to occur and that non-permissiveness is somehow due to an inability to perform membrane fusion. It remains open whether the lack of rosettes and Ca2+-ATPase activity at trichocyst attachment sites are primary cause of non-permissiveness.

scholarly journals Mg2+-binding shifts the IM30 activity from membrane protection to membrane destabilization

2020 ◽  
Author(s):  
Benedikt Junglas ◽  
Amelie Axt ◽  
Carmen Siebenaller ◽  
Hilal Sonel ◽  
Nadja Hellmann ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Thylakoid Membrane ◽  
Cellular Localization ◽  
Supported Bilayers ◽  
Force Microscopy ◽  
Membrane Surfaces ◽  
Atomic Force ◽  
Spatio Temporal ◽  
Chloroplast Stroma ◽  
External Stimuli

ABSTRACTThe inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. A soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting processes have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. The latter process depends on Mg2+-binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here we show that interaction of Mg2+ with IM30 results in increased binding of IM30 to native as well as model membranes. Via Atomic Force Microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in presence of Mg2+. The observed interaction of IM30 with membrane surfaces differs dramatically from previously observed membrane-stabilizing, carpet-like structures in the absence of Mg2+. Mg2+-induced alterations of the IM30 structure switches the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Ultrastructure of Choriocarcinoma in Vitro

1969 ◽  
Vol 27 ◽  
pp. 362-363
Author(s):  
John C. Garancis ◽  
R. A. Pattillo
Keyword(s):  
Cell Line ◽  
Physiological Function ◽  
Cell System ◽  
Bewo Cell ◽  
Bewo Cells ◽  
Gestational Choriocarcinoma ◽  
Bewo Cell Line

Growth of cell system (BeWo-cell line) derived from human gestational choriocarcinoma has been established and continuously maintained in-vitro. Furthermore, it is evident from the previous studies that this cell line has retained the physiological function of the placental trophoblasts, namely the synthesis of human chorionic gonadotrophil(HCG).The BeWo cells were relatively small and possessed single nuclei, thus indicating that this cell line consists exclusively of cytotrophoblasts. In some instances cells appeared widely separated and their lateral surfaces were provided with numerous microvilli (Fig.1).

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

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