In vitro characterization of full-length C. thermophilum Vps1 and a minimal Vps1 GG construct. (A) Growth of WT (W303A) or Δvps1 cells expressing the indicated constructs on YPD plates at 30 or 37°C. The leftmost spot in each case corresponds with 2 µl of a culture of OD₆₀₀ 0.5. Spots to the right of this correspond with 2 µl sequential fivefold dilutions. (B) Lipid nanotubes containing 40% DOPS and 20% PI3P were incubated with C. thermophilum Vps1 before staining and imaging. Top: Naked nanotube. Bottom: Nanotube decorated with Vps1. Bar, 50 nm. (C) Molecular weight determination of C. thermophilum Vps1 GG. Absolute molecular weights (shown in blue across the elution peaks and plotted on the right-hand axes) were determined using SEC-MALS. Elution peaks are plotted on the left-hand axes. Calculated molecular weights of monomers and dimers are shown with dotted red lines. (D) Kinetic analysis of the basal rate of GTP hydrolysis by C. thermophilum Vps1 GG. Initial rates of GTP hydrolysis at 37°C were plotted against the GTP concentration. The fit to the curve yielded kcat and K_M. Each point is shown as mean ± SD (n = 3).