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. 2016 Aug 1;214(3):275–291. doi: 10.1083/jcb.201603105

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© 2016 McLelland et al.

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Figure 9. — The HOPS complex acts as a tether during MDV turnover. (A) The HOPS subunit Vps41 is present on MDVs generated in vitro. Equal volumes of purified MDVs (equivalent to fraction 16 in Fig. 1 D) generated in a cell-free assay as in Fig. 1 D (with cytosol and 50 µM antimycin A), as well as material present at the bottom of the gradient (equivalent to fraction 22 in Fig. 1 D) were separated by SDS-PAGE and immunoblotted for the indicated protein. Eps15 is included as a control for soluble proteins. (B) Coimmunoprecipitation of endogenous Vps41 from COS7 cells expressing either an empty YFP vector (YFP; note that the blot is cropped above the ∼30-kD band corresponding to YFP), wild-type (WT), or mutant (Q196R) YFP-Stx17, immunoprecipitated using an anti-GFP antibody. Immunoprecipitates were separated, along with 2.5% input, by SDS-PAGE and immunoblotted for the indicated protein. (C) Coimmunoprecipitation of endogenous SNAP29 and VAMP7 from control, Vps39-, or Vps41-depleted COS7 cells expressing YFP or YFP-Stx17, immunoprecipitated using an anti-GFP antibody. Immunoprecipitates were separated, along with 2.5% input, by SDS-PAGE and immunoblotted for the indicated protein. (D) Immunoblot analysis of COS7 cells transfected with the indicated siRNA. (E) Representative confocal images of COS7 cells transfected with the indicated siRNA and LAMP1-YFP (green) that were treated with 25 µM antimycin A for 45 min, then fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). PDH E2/E3bp⁺/TOM20⁻ structures that are positive (red arrowheads) or negative (blue arrowheads) for LAMP1 are indicated. Bar, 2 µm. (F) Quantification of the percent of PDH E2/E3bp⁺/TOM20⁻ structures that colocalize with LAMP1-YFP in the cells from E. Bars represent the mean; n = 30–35 cells per condition; ***, P < 0.001. (G) Model of MDV–late endosome fusion mediated by Stx17. Stx17 is recruited to MDVs during budding, likely mobilized from a diffuse pool present on the OMM. In a HOPS-dependent manner, Stx17 forms a ternary SNARE complex with SNAP29 and VAMP7 at the late endosome to mediate fusion.