Understanding the mechanism of membrane fusion: How does the vacuolar tethering complex HOPS initiate the first contact between membranes? And by which means does it integrate SNARE, Rab, and lipidic functions in membrane fusion?
Final Report Abstract
A characteristic of eukaryotic cells is their internal organization by distinct, highly specialized, membrane-enclosed compartments. The vesicular traffic that functionally connects these individual compartments is governed by a complex machinery that facilitates the formation of vesicles with selective cargo, vesicle movement, specific tethering, and fusion of the vesicle and organelle membranes. The fusion of yeast vacuoles has been established for many years as one of the premier systems to study the process of membrane fusion. In an attempt to decipher the molecular mechanisms that allow fusion in greater detail, an ongoing effort aims to faithfully reproduce the process of vacuole fusion by in vitro reconstitution from purified components. Joining in on this effort, the objective of this project was to advance the experimental approaches that are used to dissect the many steps that contribute to the fusion process, and to analyze the fusion process with an emphasis on the role of the hexameric tethering complex HOPS (homotypic vacuole fusion and protein sorting). The fusion of proteoliposomes in vitro has classically been monitored by fluorescent lipid dequenching assays. To have more rigorous means to assess the fusion process, we have developed an assay that makes it possible to concurrently measure the mixing of membrane lipids and the protected mixing of lumenal compartments. Employing this assay we found that fusion is accompanied by a significant amount of lysis, indicating that the current reconstitution does not yet faithfully reproduce a physiological fusion event. Furthermore, and most strikingly, we found that fusion reactions that solely rely on SNARE proteins, the set of proteins that is considered to be the core machinery of fusion, are highly inefficient (as judged by the very low rate of lumenal compartment mixing). The HOPS complex facilitates fusion under these conditions by modifying the quality rather then the quantity of trans-SNARE complex interactions. We have shown that HOPS is capable of recruiting the soluble SNARE Vam7, but its role in catalyzing the formation of fusion-competent trans-SNARE complexes extends beyond that. To understand whether the interaction of the effector complex HOPS with the Rab-GTPase Ypt7 serves as a functional switch, we analyzed the affinities that HOPS exploits to fulfill its functions. Its interaction with Ypt7 is modulated by phosphorylation and also involves acidic membrane lipids. Analyzing the role of various membrane lipids, we not only found that acidic lipids can play an indirect role, but that specific lipids that are prone to form non-bilayer structures have a direct involvement in the fusion process. Future work will focus on trying to understand how HOPS catalyzes the formation of productive trans-SNARE interactions, and which additional factors/circumstances are important to achieve membrane fusion at physiological SNARE concentrations.
Publications
- (2011). Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis. Mol Biol Cell 22:4635–4646
Zucchi PC, and Zick M
(See online at https://doi.org/10.1091/mbc.E11-08-0680) - (2012). Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion. Mol Biol Cell 23:3429–3437
Zick M, and Wickner W
(See online at https://doi.org/10.1091/mbc.E12-04-0279) - (2013). The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes. Mol Biol Cell 24:3746–3753
Zick M, and Wickner W
(See online at https://doi.org/10.1091/mbc.E13-07-0419) - (2014). A distinct tethering step is vital for vacuole membrane fusion. eLIFE 3:1–19
Zick M, and Wickner WT
(See online at https://doi.org/10.7554/eLife.03251.001) - (2014). Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion. eLIFE 3:1–13
Zick M, Stroupe C, Orr A, Douville D, and Wickner WT
(See online at https://doi.org/10.7554/eLife.01879.001) - (2014). Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion. Mol Biol Cell. doi: 10.1091/mbc.E14-08-1298
Orr A, Wickner W, Rusin SF, Kettenbach AN, and Zick M
(See online at https://doi.org/10.1091/mbc.E14-08-1298)
