Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Song, Hongki; Wickner, William

doi:10.7554/elife.73240

Cited by 5 publications

(2 citation statements)

References 47 publications

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“…Content exchange could be tracked by fluorescence signals as well, but the mechanisms vary from specific reagents applied in the system ( Table 1 ). Meanwhile, simultaneous detection of lipid and content exchange in liposome fusion has been introduced in recent literature (Camacho et al 2021 ; Quade et al 2019 ; Song et al 2021 ; Song and Wickner 2021 ; Stepien et al 2019 ). Furthermore, the development of single-molecule techniques makes it possible to track single-liposome fusion events within a sub-second time scale (Bao et al 2018 ; Diao et al 2012 ; Heo et al 2021 ; Kyoung et al 2013 ; Kiessling et al 2015 ; Lai et al 2017 ).…”

Section: Development Of Snare-mediated Liposome Fusionmentioning

confidence: 99%

A practical guide for fast implementation of SNARE-mediated liposome fusion

Wang,

2024

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Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAER) family proteins are the engines of most intra-cellular and exocytotic membrane fusion pathways (Jahn and Scheller 2006 ). Over the past two decades, in-vitro liposome fusion has been proven to be a powerful tool to reconstruct physiological SNARE-mediated membrane fusion processes (Liu et al. 2017 ). The reconstitution of the membrane fusion process not only provides direct evidence of the capability of the cognate SNARE complex in driving membrane fusion but also allows researchers to study the functional mechanisms of regulatory proteins in related pathways (Wickner and Rizo 2017 ). Heretofore, a variety of delicate methods for in-vitro SNARE-mediated liposome fusion have been established (Bao et al. 2018 ; Diao et al. 2012 ; Duzgunes 2003 ; Gong et al. 2015 ; Heo et al . 2021 ; Kiessling et al. 2015 ; Kreye et al. 2008 ; Kyoung et al. 2013 ; Liu et al. 2017 ; Scott et al. 2003 ). Although technological advances have made reconstitution more physiologically relevant, increasingly elaborate experimental procedures, instruments, and data processing algorithms nevertheless hinder the non-experts from setting up basic SNARE-mediated liposome fusion assays. Here, we describe a low-cost, timesaving, and easy-to-handle protocol to set up a foundational in-vitro SNARE-mediated liposome fusion assay based on our previous publications (Liu et al. 2023 ; Wang and Ma 2022 ). The protocol can be readily adapted to assess various types of SNARE-mediated membrane fusion and the actions of fusion regulators by using appropriate alternative additives ( e . g ., proteins, macromolecules, chemicals, etc .). The total time required for one round of the assay is typically two days and could be extremely compressed into one day.

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Section: Development Of Snare-mediated Liposome Fusionmentioning

confidence: 99%

A practical guide for fast implementation of SNARE-mediated liposome fusion

Wang,

2024

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“…While absent from the Arabidopsis literature, we found evidence of such stalling and activation phenomena amongst the comparatively well-studied proteins involved in yeast vacuole fusion. Specifically, we noted the example of Sec17 (SGD:S000000146), an alpha NSF attachment protein (α-SNAP) with a multifaceted role in yeast membrane fusion [28][29][30][31][32][33]. Sec17 facilitates rapid fusion when added to mixtures of stalled intermediate complexes in reconstituted proteoliposome experiments [33,34] involving truncated SNARE proteins which cannot fully zipper together to drive membrane fusion.…”

Section: Conceptual Model Suggests a Missing Signaling Event And Yeas...mentioning

confidence: 99%

Model-based inference of a plant-specific dual role for HOPS in regulating guard cell vacuole fusion

Hodgens,

Flaherty,

Pullen

et al. 2023

Preprint

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Stomata are the pores on a leaf surface that regulate gas exchange. Each stoma consists of two guard cells whose movements regulate pore opening and thereby control CO2fixation and water loss. Guard cell movements depend in part on the remodeling of vacuoles, which have been observed to change from a highly fragmented state to a fused morphology during stomata opening. This change in morphology requires a membrane fusion mechanism that responds rapidly to environmental signals, allowing plants to respond to diurnal and stress cues. With guard cell vacuoles being both large and responsive to external signals, stomata represent a unique system in which to delineate mechanisms of membrane fusion.Fusion of vacuole membranes is a highly conserved process in eukaryotes, with key roles played by two multi-subunit complexes: HOPS (homotypic fusion and vacuolar protein sorting) and SNARE (soluble NSF attachment protein receptor). HOPS is a vacuole tethering factor that is thought to chaperone SNAREs from apposing vacuole membranes into a fusion-competent complex capable of rearranging membranes. To resolve a counter-intuitive observation regarding the role of HOPS in regulating plant vacuole morphology, we derived a quantitative model of vacuole fusion dynamics and used it to generate testable predictions about HOPS-SNARE interactions. We derived our model by applying simulation-based inference to integrate prior knowledge about molecular interactions with limited, qualitative observations of emergent vacuole phenotypes. By constraining the model parameters to yield the emergent outcomes observed for stoma opening – as induced by two distinct chemical treatments – we predicted a dual role for HOPS and identified a stalled form of the SNARE complex that differs from phenomena reported in yeast. We predict that HOPS has contradictory actions at different points in the fusion signaling pathway, promoting the formation of SNARE complexes, but limiting their activity.Author summaryPlants “breathe” through pores in their leaves where each pore is formed by two specialized cells called guard cells. To open these pores, guard cells change in volume. This volume change is controlled by water-filled organelles called vacuoles that morph from multiple small entities to a few large ones capable of taking up more water to reshape the cell. Specialized proteins in vacuole membranes make this change happen by pulling vacuoles together until they fuse. Some of these proteins reside in membranes, but others must be drawn to the membrane from the cell’s cytoplasm. Specific lipid molecules in the membrane play an important role in recruiting those proteins to the vacuole membrane. We previously made an unexpected finding that removing this lipid induces plant vacuole fusion. To make sense of this observation, we used a mathematical model to piece together our knowledge of the proteins involved in this process and what we know about the chemical treatments that cause vacuoles to morph. Using computer simulations, we uncovered new rules about how molecules interact in membranes to accomplish the task of vacuole fusion in plants. We think the rules uncovered through mathematical modeling allow plants to respond quickly to environmental cues.

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Pathway engineering facilitates efficient protein expression in Pichia pastoris

Liu

Gong

et al. 2022

Appl Microbiol Biotechnol

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Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS

Cited by 5 publications

References 47 publications

A practical guide for fast implementation of SNARE-mediated liposome fusion

A practical guide for fast implementation of SNARE-mediated liposome fusion

Model-based inference of a plant-specific dual role for HOPS in regulating guard cell vacuole fusion

Pathway engineering facilitates efficient protein expression in Pichia pastoris

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