Phosphatidylserine Inhibits and Calcium Promotes Model Membrane Fusion

Tarafdar, Pradip K.; Chakraborty, Hirak; Dennison, S. Moses; Lentz, Barry R.

doi:10.1016/j.bpj.2012.09.030

Cited by 35 publications

(48 citation statements)

References 37 publications

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“…The latter includes Ca 2+ -triggered insertion of synaptotagmin at the neuronal synapse (48) and Sec17 for intracellular fusion. In model systems, transition from apposed bilayers to fusion has even been promoted by interaction of calcium with lipids (49). The relative contribution of each to achieving the activation state for transition to fusion remains for future biophysical studies; important progress has been made in this regard with SNAREs (50).…”

Section: Discussionmentioning

confidence: 99%

Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

Zick

Orr

Schwartz³

et al. 2015

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

110

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Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP-or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17 L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17 F21S,M22S , with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17 F21S,M22S , interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.I ntracellular vesicular traffic between organelles is the basis of cell growth, hormone secretion, and neurotransmission. At each step of exocytic and endocytic trafficking, membranes dock and fuse, mixing their lipids and luminal contents while keeping them separate from the cytosol. Families of proteins, conserved from yeast to humans, mediate docking and fusion. Fusion requires Rab family GTPases and "effector" proteins that bind to a Rab in its active, GTP-bound state (1). Among the effectors are large, organelle-specific tethering complexes. Fusion requires SNARE proteins and their chaperones. SNAREs (2) are proteins that can "snare" (bind to) each other, in cis (when anchored to the same membrane) or in trans (when anchored to apposed, tethered membranes). SNAREs have a conserved "SNARE domain" with a characteristic heptad repeat. SNAREs are categorized as R-SNAREs if they have a central arginyl residue, or Qa-, Qb-, or Qc-SNAREs with a central glutamyl residue (3). SNAREs form RQaQbQc quaternary cis-or trans-SNARE complexes, which bind SNARE chaperones, including the Sec1/Munc18 family of SNARE binding proteins, and Sec18/NSF (N-ethylmaleimidesensitive factor), an AAA family ATPase that drives SNARE c...

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Section: Discussionmentioning

confidence: 99%

Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

Zick

Orr

Schwartz³

et al. 2015

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

110

View full text Add to dashboard Cite

show abstract

“…We have found that a sequential, two-intermediate model for fusion is able to account for the ensemble kinetics of the fusion process for a variety of membrane systems (19,22,31,32) over a range of temperatures (19). Our ensemble kinetic treatment is based on the expanded stalk structural model for fusion (33) as modified to treat the highly curved, space-encapsulating membranes of SUVs (17).…”

Section: Discussionmentioning

confidence: 99%

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

Sengupta

Chakraborty

Lentz

2014

Biophysical Journal

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We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2 state) that converts to a final fusion pore state with a combined rate k3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate microstructures.

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“…We used an EDTA-buffered Ca 2þ system to ensure that Ca 2þ leached from the glassware would not add uncertainty to our results. Because the complexation constant for Ca 2þ by EDTA is so large (K ¼ [Ca 4 EDTA]/[Ca]][EDTA] varies from~10 4 at pH 5 to~10 8 at pH 8), the amount of free Ca 2þ is determined by the error limits of the measurements that are required to prepare the buffer and does not vary with pH or temperature (21). is a thermodynamic ensemble of similar molecular arrangements (microstructures) (24).…”

Section: Vesicle Preparationmentioning

confidence: 99%

pH Alters PEG-Mediated Fusion of Phosphatidylethanolamine-Containing Vesicles

Chakraborty

Sengupta

Lentz

2014

Biophysical Journal

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Here, we examine the different mechanisms of poly(ethylene glycol)-mediated fusion of small unilamellar vesicles composed of dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamine (DOPE)/sphingomyelin/cholesterol in a molar ratio of 35:30:15:20 at pH 7.4 versus pH 5. In doing so, we test the hypothesis that fusion of this lipid mixture should be influenced by differences in hydration of DOPE at these two pH values. An examination of the literature reveals that DOPE should be less hydrated at pH 5 (where influenza virus particles fuse with endosome membranes) than at pH 7.4 (where synaptic vesicles or HIV virus particles fuse with plasma membrane). Ensemble kinetic experiments revealed substantial differences in fusion of this plasma membrane mimetic system at these two pH values. The most dramatic difference was the observation of two intermediates at pH 5 but loss of one of these fusion intermediates at pH 7.4. Analysis of data collected at several temperatures also revealed that formation of the initial fusion intermediate (stalk) was favored at pH 7.4 due to increased activation entropy. Our observations support the hypothesis that the different negative intrinsic curvature of DOPE can account for different fusion paths and activation thermodynamics in steps of the fusion process at these two pH values. Finally, the effects of 2 mol % hexadecane on fusion at both pH values seemed to have similar origins for step 1 (promotion of acyl chain or hydrocarbon excursion into interbilayer space) and step 3 (reduction of interstice energy leading to expansion to a critical stalk radius). Different hexadecane effects on activation thermodynamics at these two pH values can also be related to altered DOPE hydration. The results support our kinetic model for fusion and offer insight into the critical role of phosphatidylethanolamine in fusion.

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Phosphatidylserine Inhibits and Calcium Promotes Model Membrane Fusion

Cited by 35 publications

References 37 publications

Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

Sec17 can trigger fusion of trans -SNARE paired membranes without Sec18

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

pH Alters PEG-Mediated Fusion of Phosphatidylethanolamine-Containing Vesicles

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