Figure 1.

Gradient fractionation of post-Golgi SVs accumulated in 38°C-shifted sec6-4 cells. Membranes enriched in SVs were prepared from 38°C-shifted or 27°C-grown sec6-4 cells by differential centrifugation, loaded on the bottom of a linear Nycodenz/sorbitol gradient, and then floated to equilibrium by centrifugation. (A) Fractions were collected from the top and analyzed for enzyme activities and by immunoblotting. Enzyme activities were determined as described in Materials and Methods and expressed in arbitrary units (a.u.) based on the absorbance measured at 660 nm (PM-ATPase) or 540 nm (invertase). Immunoblots were probed with a mAb against the HA epitope to detect HA-tagged Pma1p. (B) Lipid phosphorus content per fraction was determined as described in Materials and Methods. (C) Fraction densities were measured by reading refractive indices on a Bausch & Lomb refractometer.