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. 2012 Feb 1;2(2):59–69. doi: 10.4161/bioa.20359

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Copyright © 2012 Landes Bioscience

This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.

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Figure 3. — A HOPS dimer catalyzes Ypt7-dependent trans QbQcR-Qa SNARE complex formation. (A) trans-SNARE complexes assayed by tagged SNAREs. Vam3-HA vacuoles from the wild type strain were mixed with Nyv1-VSV vacuoles from either the wild type strain (lanes 1 and 2) or the Ypt7-T22N strain (lanes 3 and 4) and incubated with or without ATP under standard fusion conditions. Vam3-HA was precipitated using Protein-G/antiHA antibody and co-precipitating Nyv1-VSV was detected by SDS-PAGE and western blotting with antiNyv1 antibody. A trans-SNARE complex is observed only in the presence of ATP when both partners are wild type (lane 2) but not in a Rab mutant background for one of the partners (lane 4). The panel on the right side compares fusion efficiency between both wild type partners with that between the wild type and Ypt7-T22N mutant quantified and normalized from three independent experiments (p < 0.001). (B) trans-HOPS/Qa SNARE interactions. Vacuoles from the ΔN-terminal Vam3 strain harboring Vps16-HA were mixed with vacuoles from either the wild type strain (lanes 1 and 2) or the Ypt7-T22N strain (lanes 3 and 4) and incubated with or without ATP under standard fusion conditions. Vps16-HA was precipitated using Protein-G/antiHA antibody and co-precipitating Vam3 was detected by SDS-PAGE and western blotting with antiVam3 antibody. Vam3 (trans-Qa SNARE) co-precipitates with Vps16-HA only in presence of ATP when both partners are wild type (lane 2) but not in a Rab mutant background for one of the partners (lane 4). Lanes 5–8 show corresponding protein inputs. (C) Vacuoles from the ΔN-terminal Vam3 strain harboring Vps16-HA were mixed with vacuoles from the Nyv1-VSV strain and incubated with ATP alone (lane 1) or both ATP and GDI (lane 2) under standard fusion conditions. Vps16-HA was precipitated using Protein-G/antiHA antibody and co-precipitating SNAREs were analyzed by SDS-PAGE and western blotting against the indicated proteins. Full length Vam3 (trans Qa SNARE) and Nyv1 (cis R SNARE) co-precipitate with Vps16-HA in an ATP-dependent manner as. Lane 3 shows corresponding protein inputs.