Figure 6.

The membrane association of Vac1HAp is not dependent on the functions of Pep12p, Vps34p, Vps21p, Vps45p, or the Vac1p FYVE finger. The subcellular fractionation patterns of Vac1HAp were assessed in the following strains that expressed Vac1HAp from a CEN plasmid; GTY104 (WT, panels 1 and 2), GTY108 (Δpep12, panel 3), GTY109 (Δvps34, panel 4), GTY111 (Δpep12Δvps34, panel 5), GTY106, (Δvps21, panel 6), and GTY107 (Δvps45, panel 7) and that expressed C221/292S-Vac1HAp from a CEN plasmid, GTY104 (C221/292S-Vac1HAp, panel 8). Spheroplasts (20 OD₆₀₀ equivalents) of these strains were lysed, and unbroken cells were cleared by centrifugation at 500 × g. Five OD₆₀₀ equivalents of these supernatants were then centrifuged at 13,000 × g to generate a P13 pellet and an S13 supernatant. Five OD₆₀₀ equivalents of the S13 were further fractionated by spinning at 100,000 × g to generate a P100 pellet and an S100 supernatant. Supernatants were precipitated with TCA and resuspended in urea sample buffer as described. Pellets were directly resuspended in urea sample buffer. All fractions were resolved by SDS-PAGE, and Vac1HA proteins were identified by Western blot analysis using HA antibodies. Vac1HAp and C221/292S-Vac1HAp were visualized using Blaze chemilluminescent detection reagents.