Biomedical subjects

Yeon-Kyun Shin

Publications and source records attributed to Yeon-Kyun Shin.

18 recordsLinked to original sources

PubMed2006

Multiple intermediates in SNARE-induced membrane fusion.

Membrane fusion in eukaryotic cells is thought to be mediated by a highly conserved family of proteins called SNAREs (soluble N-ethyl maleimide sensitive-factor attachment protein receptors). The vesicle-associated v-SNARE engages with its partner t-SNAREs on the target membrane to form a coiled coil that bridges two membranes and facilitates fusion. As demonstrated by recent findings on the hemifusion state, identifying intermediates of membrane fusion can help unveil the underlying fusion mechanism. Observation of SNARE-driven fusion at the single-liposome level has the potential to dissect and characterize fusion intermediates most directly. Here, we report on the real-time observation of lipid-mixing dynamics in a single fusion event between a pair of SNARE-reconstituted liposomes. The assay reveals multiple intermediate states characterized by discrete values of FRET between membrane-bound fluorophores. Hemifusion, flickering of fusion pores, and kinetic transitions between intermediates, which would be very difficult to detect in ensemble assays, are now identified. The ability to monitor the time course of fusion events between two proteoliposomes should be useful for addressing many important issues in SNARE-mediated membrane fusion.

Fluorescence Resonance Energy Transfer↗

PubMed2006

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo.

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.

Amino Acid Motifs↗

PubMed2006

Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I.

Synaptic transmission relies on an exquisitely orchestrated series of protein-protein interactions. Here we show that fusion driven by neuronal SNAREs is inhibited by the regulatory protein complexin. Furthermore, inner-leaflet mixing is strongly impaired relative to total lipid mixing, indicating that inhibition by complexin arrests fusion at hemifusion. When the calcium sensor synaptotagmin is added in the presence of calcium, inhibition by complexin is relieved and full fusion rapidly proceeds.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Membrane topology of helix 0 of the Epsin N-terminal homology domain.

Specific interaction of the epsin N-terminal homology (ENTH) domain with the plasma membrane appears to bridge other related proteins to the specific regions of the membrane that are invaginated to form endocytic vesicles. An additional a-helix, referred to as helix 0 (H0), is formed in the presence of the soluble ligand inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] at the N terminus of the ENTH domain (amino acid residues 3-15). The ENTH domain alone and full-length epsin cause tubulation of liposomes made of brain lipids. Thus, it is believed that H0 is membrane-inserted when it is coordinated with the phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], resulting in membrane deformation as well as recruitment of accessory factors to the membrane. However, formation of H0 in a real biological membrane has not been demonstrated. In the present study, the membrane structure of H0 was determined by measurement of electron paramagnetic resonance (EPR) nitroxide accessibility. H0 was located at the phosphate head-group region of the membrane. Moreover, EPR line-shape analysis indicated that no pre-formed H0-like structure were present on normal acidic membranes. PtdIns(4,5)P2 was necessary and sufficient for interaction of the H0 region with the membrane. H0 was stable only in the membrane. In conclusion, the H0 region of the ENTH domain has an intrinsic ability to form H0 in a PtdIns(4,5)P2-containing membrane, perhaps functioning as a sensor of membrane patches enriched with PtdIns(4,5)P2 that will initiate curvature to form endocytic vesicles.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Transmembrane organization of yeast syntaxin-analogue Sso1p.

Membrane fusion in secretory pathways is thought to be mediated by SNAREs. It is proposed that membrane fusion transits through hemifusion, a condition in which the outer leaflets of the bilayers are mixed, but the inner leaflets are not. Hemifusion then proceeds to the fusion pore that connects the two internal contents. It is believed that the transmembrane domains (TMDs) of the fusion proteins play an essential role in the transition from hemifusion to the fusion pore. In this work, the structure, dynamics, and membrane topology of the TMD of Sso1p, a target membrane (t-) SNARE involved in the trafficking from Golgi to plasma membrane in yeast, was investigated using site-directed spin labeling and EPR spectroscopy. The EPR analysis of spin-labeled mutants showed that the TMD of Sso1p is a well-defined membrane spanning alpha-helix. The results also indicate that there is an equilibrium between the monomers and the oligomers. The oligomerization is mainly mediated through the interaction at the N-terminal half of the TMD, whereas the C-terminal half is free of the tertiary interaction. Additionally, the isotropic hyperfine splitting values were examined for nitroxide-scanning mutants, and it was found that the hyperfine splitting values show a V-shaped profile across the bilayer. Thus, hyperfine splitting may be used as an additional parameter to measure bilayer immersion depths of nitroxide.

Amino Acid Sequence↗

PubMed2006

Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion.

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca(2+). Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca(2+)-dependent manner. Syt.Ca(2+) increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt.Ca(2+) was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt.Ca(2+) stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt.Ca(2+) was required for the stimulation of fusion.

Animals↗

PubMed2005

YKT6 is a core constituent of membrane fusion machineries at the Arabidopsis trans-Golgi network.

SNARE complex formation is essential for membrane fusion in exocytotic and vacuolar trafficking pathways. Vesicle-associated (v-) SNARE associates with a target membrane (t-) SNARE to form a SNARE complex bridging two membranes, which may facilitate membrane fusion. The Arabidopsis genome encodes a large number of predicted SNARE proteins that might function primarily as fusogens for vesicle transport in endomembrane systems. The SNAREs SYP41, SYP61 and VTI12 reside in the trans-Golgi network and have been proposed to function together in vesicle fusion with this organelle. Here, we use a liposome fusion assay to demonstrate that VTI12 and either SYP41 or SYP61, but not both, are required for membrane fusion. This indicates that SYP41 and SYP61 are likely to function in independent vesicle fusion reactions in Arabidopsis. In addition, we have identified two new functionally interchangeable components, YKT61 and YKT62, that show sequence similarity to the multifunctional yeast SNARE YKT6. Both YKT61 and YKT62 interact with SYP41 and are essential for membrane fusion mediated by either SYP41 or SYP61. These results therefore define the core constituents required for membrane fusion at the Arabidopsis trans-Golgi network.

Arabidopsis↗

PubMed2005

Membrane fusion induced by neuronal SNAREs transits through hemifusion.

Synaptic transmission requires the controlled release of neurotransmitter from synaptic vesicles by membrane fusion with the presynaptic plasma membrane. SNAREs are the core constituents of the protein machinery responsible for synaptic membrane fusion. The mechanism by which SNAREs drive membrane fusion is thought to involve a hemifusion intermediate, a condition in which the outer leaflets of two bilayers are combined and the inner leaflets remain intact; however, hemifusion has been observed only as an end point rather than as an intermediate. Here, we examined the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs using fluorescence assays that monitor both total lipid mixing and inner leaflet mixing. Our results demonstrate that hemifusion is dominant at the early stage of the fusion reaction. Over time, hemifusion transitioned to complete fusion, showing that hemifusion is a true intermediate. We also show that hemifusion intermediates can be trapped, likely as unproductive outcomes, by modulating the surface concentration of the SNARE proteins.

Animals↗

PubMed2005

Hemifusion in SNARE-mediated membrane fusion.

SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wild-type SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate.

Electron Spin Resonance Spectroscopy↗

PubMed2005

A partially zipped SNARE complex stabilized by the membrane.

The SNARE complex acts centrally for intracellular membrane fusion, an essential process for vesicular transport in cells. Association between vesicle-associated (v-) SNARE and target membrane (t-) SNARE results in the coiled coil core that bridges two membranes. Here, the structure of the SNARE complex assembled by recombinant t-SNARE Sso1p/Sec9 and v-SNARE Snc2p, which are involved in post-Golgi trafficking in yeast, was investigated using EPR. In detergent solutions, SNAREs formed a fully assembled core. However, when t-SNAREs were reconstituted into the proteoliposome and mixed with the soluble SNARE motif of Snc2p, a partially zipped core in which the N-terminal region is structured, whereas the C-terminal region is frayed, was detected. The partially zipped and fully assembled complexes coexisted with little free energy difference between them. Thus, the core complex formation of yeast SNAREs might not serve as the energy source for the fusion, which is different from what has been known for neuronal SNAREs. On the other hand, the results from the proteoliposome fusion assay, employing cysteine- and nitroxide-scanning mutants of Sso1p, suggested that the formation of the complete core is required for membrane fusion. This implies that core SNARE assembly plays an essential role in setting up the proper geometry of the lipid-protein complex for the successful fusion.

Amino Acid Sequence↗

PubMed2004

SNARE assembly and membrane fusion, a kinetic analysis.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) assembly may promote intracellular membrane fusion, an essential process for vesicular transport in cells. Core complex formation between vesicle-associated SNARE and target membrane SNARE perhaps drives the merging of two membranes into a single bilayer. Using spin-labeling EPR, trans-SNARE complex formation was monitored "locally" at four different core locations of recombinant yeast SNAREs, which are individually reconstituted into phospholipid vesicles. The results indicate that the time scales of core formation are virtually the same at all four locations throughout the core region, indicating the possibility of a single step core assembly, which appears to be somewhat different from what has been postulated by the "zipper" model. The EPR data were then compared with the kinetics of the lipid mixing measured with the fluorescence assay. The analysis suggests that SNARE core assembly occurs on a much faster time scale than the lipid mixing, providing a new insight into the timing of individual events in SNARE-induced membrane fusion.

Cell Membrane↗

PubMed2004

Constitutive versus regulated SNARE assembly: a structural basis.

SNARE complex formation is essential for intracellular membrane fusion. Vesicle-associated (v-) SNARE intertwines with target membrane (t-) SNARE to form a coiled coil that bridges two membranes and facilitates fusion. For the SNARE family involved in neuronal communications, complex formation is tightly regulated by the v-SNARE-membrane interactions. However, it was found using EPR that complex formation is spontaneous for a different SNARE family that is involved in protein trafficking in yeast. Further, reconstituted yeast SNAREs promoted membrane fusion, different from the inhibited fusion for reconstituted neuronal SNAREs. The EPR structural analysis showed that none of the coiled-coil residues of yeast v-SNARE is buried in the hydrophobic layer of the membrane, making the entire coiled-coil motif accessible, again different from the deep insertion of the membrane-proximal region of neuronal v-SNARE into the bilayer. Importantly, yeast membrane fusion is constitutively active, while synaptic membrane fusion is regulated, consistent with the present results for two SNARE families. Thus, the v-SNARE-membrane interaction may be a major molecular determinant for regulated versus constitutive membrane fusion in cells.

Amino Acid Sequence↗

PubMed2003

Insertion of the membrane-proximal region of the neuronal SNARE coiled coil into the membrane.

In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an alpha-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling.

Electron Spin Resonance Spectroscopy↗

PubMed2003

Regulation of neuronal SNARE assembly by the membrane.

In the neuron, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) assembly acts centrally in driving membrane fusion, a required process for neurotransmitter release. In the cytoplasm, vesicular SNARE VAMP-2 (vesicle-associated membrane protein-2) engages with two plasma membrane SNAREs, syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa), to form the core complex that bridges two membranes. Although various factors regulate SNARE assembly, the membrane also aids in regulation by trapping VAMP-2 in the membrane. Fluorescence and EPR analyses revealed that the insertion of seven C-terminal core-forming residues into the membrane controls complex formation of the entire core region, even though the preceding 54 core-forming residues are fully exposed and freely moving. When two interfacial tryptophan residues in this region were replaced with hydrophilic serine residues, the mutation supported rapid complex formation. The results suggest that the membrane-proximal region of VAMP-2 is a regulatory module for SNARE assembly, providing new insights into calcium-triggered membrane fusion.

Antigens, Surface↗

PubMed2002

Membrane topologies of neuronal SNARE folding intermediates.

Assembly of the SNARE complex is essential for neurotransmitter release at synapses. Target plasma membrane SNAREs (t-SNAREs) syntaxin 1A and SNAP-25 form the t-SNARE complex that serves as an intermediate toward final SNARE assembly with vesicle-associated SNARE (v-SNARE). Membrane topologies of syntaxin 1A and the t-SNARE complex were investigated using site-directed spin labeling EPR. EPR analysis revealed that the basic region at the membrane-water interface is unstructured but inserted into the membrane. Such membrane insertion leaves no gap between the t-SNARE core and the membrane. Yet the lack of structure could provide the flexibility necessary for the t-SNARE core. Further, the insertion of the basic interfacial region into the membrane may have profound implications for the mechanism of SNARE-induced membrane fusion.

Amino Acid Sequence↗

PubMed2002

The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study.

The formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is an essential process for membrane fusion and the neurotransmitter release in neurons. As an initial step toward the determination of the membrane topology of the SNARE complex, residues at the membrane-water interface were investigated with site-specific spin labeling electron paramagnetic resonance. EPR analysis revealed that the basic amino acid-rich interfacial region, which is universal for all transmembrane SNARE proteins, inserts into the membrane, eliminating the gap between the core complex and the membrane. The result raises the possibility that core complex formation directly leads to the apposition of two membranes, which could facilitate membrane fusion.

Amino Acid Sequence↗

PubMed2002

Probing domain swapping for the neuronal SNARE complex with electron paramagnetic resonance.

Highly conserved soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins control membrane fusion at synapses. The target plasma membrane-associated SNARE proteins and the vesicle-associated SNARE protein assemble into a parallel four-helix bundle. Using a novel EPR approach, it is found that the SNARE four-helix bundles are interconnected via domain swapping that is achieved by substituting one of the two SNAP-25 helices with the identical helix from the second four-helical bundle. Domain swapping is likely to play a role in the multimerization of the SNARE complex that is required for successful membrane fusion. The new EPR application employed here should be useful to study other polymerizing proteins.

Cyclic N-Oxides↗

PubMed2002

The four-helix bundle of the neuronal target membrane SNARE complex is neither disordered in the middle nor uncoiled at the C-terminal region.

Assembly of the SNARE complex is an essential step for membrane fusion and neurotransmitter release in neurons. The plasma membrane SNAREs syntaxin 1A and SNAP-25 (t-SNAREs) and the delivery-vesicle SNARE VAMP2 (or v-SNARE) contain the "SNARE regions" that essentially mediate SNARE pairing. Using site-directed spin labeling and EPR distance measurement we show that two identical copies of the SNARE region from syntaxin 1A intertwine as a coiled coil near the "ionic layer" region. The structure of the t-SNARE complex appears to be virtually identical to that of the ternary SNARE complex, except that VAMP2 is substituted to the second copy of syntaxin 1A. Furthermore, it appears that the coiled coil structure is maintained up to residue 259 of syntaxin 1A, identical to that of the ternary complex. These results are somewhat contradictory to the previous reports, suggesting that the t-SNARE complex has the disordered midsection (Xiao, W. Z., Poirier, M. A., Bennett, M. K., and Shin, Y. K. (2001) Nat. Struc. Biol. 8, 308-311) and the uncoiled C-terminal region (Margittai, M., Fasshauer, D., Pabst, S., Jahn, R., and Langen, R. (2001) J. Biol. Chem. 276, 13169-13177). The newly refined structure of the t-SNARE complex provides a basis for the better understanding of the SNARE assembly process. It also provides possible structural-functional clues to the membrane fusion in the v-SNARE deleted fusion models.

Amino Acid Sequence↗