Reference: Orr A and Wickner W (2023) MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion. Mol Biol Cell mbcE23060228

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Abstract


Yeast vacuolar HOPS tethers membranes, catalyzes trans-SNARE assembly between R- and Q-SNAREs, and shepherds SNAREs past early inhibition by Sec17. After partial SNARE zippering, fusion is driven slowly by either completion of SNARE zippering or by Sec17/Sec18, but rapid fusion needs zippering and Sec17/Sec18. Using reconstituted vacuolar fusion, we find that MARCKS Effector Domain (MED) peptide, a lipid ligand, blocks fusion reversibly at a late reaction stage. The MED fusion blockade is overcome by either salt extraction, inactivation with the MED ligand calmodulin, or addition of Sec17/Sec18. During incubation with MED, SNAREs assemble stable complexes in trans and fusion becomes resistant to antibody to the Qa SNARE. When Q-SNAREs are pre-assembled, a synthetic tether can replace HOPS for fusion. With a synthetic tether, fusion needs both complete SNARE zippering and Sec17/Sec18 to overcome a MED block. In contrast, when SNARE domains are only 2/3 zippered, only HOPS will support Sec17/Sec18 driven fusion without needing complete zippering. HOPS thus remains engaged with SNAREs during zippering. MED facilitates the study of distinct fusion stages: tethering, initial trans-SNARE assembly and its sensitivity to Sec17, SNARE zippering, Sec17/Sec18 engagement, and lipid and lumenal mixing.

Reference Type
Journal Article
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Orr A, Wickner W
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