The V‐ATPase proteolipid cylinder promotes the lipid‐mixing stage of SNARE‐dependent fusion of yeast vacuoles

B Strasser, J Iwaszkiewicz, O Michielin… - The EMBO journal, 2011 - embopress.org
B Strasser, J Iwaszkiewicz, O Michielin, A Mayer
The EMBO journal, 2011embopress.org
The V‐ATPase V0 sector associates with the peripheral V1 sector to form a proton pump. V0
alone has an additional function, facilitating membrane fusion in the endocytic and late
exocytic pathways. V0 contains a hexameric proteolipid cylinder, which might support fusion
as proposed in proteinaceous pore models. To test this, we randomly mutagenized
proteolipids. We recovered alleles that preserve proton translocation, normal SNARE
activation and trans‐SNARE pairing but that impair lipid and content mixing. Critical …
The V‐ATPase V0 sector associates with the peripheral V1 sector to form a proton pump. V0 alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V0 contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans‐SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion‐impairing proteolipid substitutions stabilize the interaction of V0 with V1. Deletion of the vacuolar v‐SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V0. Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE‐dependent conformational change in V0 proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.
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