Hyperactive forms of Sly1 tether high-curvature vesicles to immobilized Sed5. (A) The ability of Sed5-bound Sly1 to directly tether vesicles was tested using a bead-based assay system. GST-Sed5 was adsorbed to glutathione-sepharose (GSH) beads, and wild-type or mutant forms of Sly1 were added to the reaction mixture. After 5 min, Texas Red-DHPE labeled liposomes were added to the mixtures, incubated for 15–20 min, and imaged by confocal microscopy (10× objective). A false-color scale was chosen to emphasize small differences in contrast under conditions with less tethering. The micrographs are representative of at least two independent assays per condition. (B–D) To quantify tethering efficiency, we used a spin-down assay. Binding reactions set up as for microscopy were subjected to low-speed centrifugation to sediment the GSH beads and associated proteins and vesicles. The supernatant was discarded and detergent was added to the pellet to liberate bound fluorescent lipids; the resulting signal was quantified by fluorometry. In C, Sly1-20 was present for each condition. In D, Sly1* was preincubated with a 6:1 excess of soluble Sed5-Habc or Sed5-N-Habc, as indicated. Y-axes show bead-associated fluorescence (au, arbitrary units) after subtracting background from blanks containing only buffer. Bars indicate means ±95% confidence intervals for 4–10 independent experiments. Binding of the Sly1 variants to immobilized Sed5 was efficient and nearly stoichiometric (Fig. S5).