Fluorescent lipids

PI3P was from Echelon (Salt Lake City, Utah), ergosterol from Sigma (St. Louis, MO), fluorescent lipids from Thermo Fisher (Waltham, MA), and other lipids were from Avanti (Alabaster, AL). Biobeads SM2 were from BioRad, Cy5-Streptavidin from SeraCare (Milford, MA), biotinylated phycoerythrin from Invitrogen (Eugene, OR), and underivatized streptavidin from Thermo Fisher. Spectrapor six dialysis tubing (7.5 mm diameter, 25 kDa cutoff) was from Spectrum Labs (Las Vegas, NV). Octyl-b-D-glucopyranoside was purchased from Anatrace (Maumee, OH).

Sec17, Sec17^FM>SS, and Qc-SNAREs were expressed in E. coli and purified as described (Schwartz and Merz, 2009 (link)). A two-tag strategy was employed. An N-terminal polyhistidine tag and a C-terminal, self-cleaving intein and chitin-binding domain tag, allowed retrieval of Qc proteins uncontaminated by N- or C-terminal cleavage products. Sec18 was expressed in E. coli and purified as described (Mayer et al., 1996 (link)). Full-length Vam3, Vti1 and Nyv1 were expressed in E. coli and purified as described (Mima et al., 2008 (link); Zick et al., 2015 (link); Zucchi and Zick, 2011 (link)). Ypt7 and HOPS were overproduced in yeast and purified as described (Zick and Wickner, 2013 (link)). Cy5-strepavidin was purchased from KPL, unlabeled avidin from Thermo Scientific (Bothell, Washington, USA), and R-phycoerythrin-biotin from Life Technologies. Monoclonal antibodies against ALP (Pho8), CPY (Prc1) and PGK1 were purchased from Molecular Probes (Life Technologies, Carlsbad, California, USA). Affinity-purified antibodies against Vam3, Vam7, Nyv1, Vti1, Sec17, Sec18, and Vps33 were prepared as described (Schwartz and Merz, 2009 (link)). Lipids were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA), except for ergosterol (Sigma-Aldrich, Saint Louis, Missouri, USA) and fluorescent lipids (Life Technologies).