Fig. 1. Membrane association of Vtc proteins. (A) Vtc1p and Vtc4p behave as integral membrane proteins. 60 µg of vacuoles from strain OMY1 in 1 ml of PS were centrifuged (10,000 g for 5 minutes at 4°C) and resuspended in 0.2 ml PS with one of the following additions: 100 mM KCl, 50 mM KOAc (low salt); 1.6 M KCl (high salt); 4 M urea; or 0.1 M Na₂CO₃. After 10 minutes at 30°C (or 30 minutes on ice for carbonate extraction), the samples were centrifuged (125,000 g, 20 minutes, 4°C). Pellets (P) were resuspended in 1 ml of PS and the supernatants (S) supplemented with PS ad 1 ml. All samples were TCA precipitated and analyzed by SDS-PAGE and western blotting. (B) Strains BJ3505 (pep4^–) and DKY6281 (PEP4⁺) expressing a Vtc1p-GFP fusion were grown logarithmically in YPD and viewed under a confocal fluorescence microscope. Left panel: GFP fluorescence; right panel: Nomarski optics.