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A Reductionist Approach to Understanding Membrane Fusion
Author(s) -
Wickner William T.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.98.1
Subject(s) - lipid bilayer fusion , rab , snare complex , microbiology and biotechnology , biology , gtpase , vacuole , vesicular transport proteins , biochemistry , fusion mechanism , organelle , protein subunit , chemistry , membrane , cytoplasm , vacuolar protein sorting , gene
Membrane fusion is essential for intracellular protein targeting, cell growth, neurotransmission, and hormone secretion. Since the mechanism of fusion is conserved from yeast to humans, we study fusion of the vacuole (lysosome) of the biochemically and genetically tractable yeast S. cerervisiae. Vacuole fusion requires phosphoinositides and non‐bilayer‐prone lipids, a Rab family GTPase, four SNARE proteins which form complexes together in cis (bound to the same membrane) or trans (bound to apposed membranes before fusion), the SNARE complex disassembly chaperone Sec18 (NSF), its cochaperone and fusion‐trigger protein Sec17, and a large, multisubunit complex termed HOPS ( ho motypic fusion and vacuole p rotein s orting). HOPS binds two membrane‐anchored Rabs to tether the organelle, catalyzes trans‐SNARE complex assembly through its Sec1/Munc18 (SM)‐homolog subunit, and directly associates with acidic lipids and phosphoinositides. Each of these factors has been shown through genetics to be required for fusion in vivo, and in vitro for fusion of the isolated organelle. To understand the mechanism, we have reconstituted fusion with each of these purified, recombinant proteins and 8 defined lipids, revealing novel aspects of membrane fusion: 1. The lipid bilayer isn't just a passive player. Acidic lipids, non‐bilayer prone lipids, phosphoinositides, and the fluidity of the fatty acyl phase are each crucial. 2. Tethering is an essential step for fusion, even where trans‐SNARE complexes have formed. 3. SNARE complex assembly is catalyzed by SM proteins and associated proteins, 4. Sec17 (a‐SNAP) binds to the trans‐SNARE complex and inserts an apolar loop into the bilayer to trigger fusion. 5. A supercomplex of HOPS with trans‐SNARE complexes is found on the purified vacuole and can be reconstituted with purified components; we are seeking its precise function in the fusion cascade. Support or Funding Information This work was supported by NIH grant GM23377‐40