(A) Soluble vacuolar alkaline phosphatase (sPho8) activity in WT and ESCRT mutants (vps36∆; vps23∆). Data are represented as fold increase in activity after 3 hr starvation over rich growth condition (n = 3; mean ± SEM). (B) Fluorescence microscopy of CPY(1–50)-mRFP and Mup1-GFP in WT cells, vps4∆ mutants and vps4∆ mutants over-expressing Vps10 growing under rich or starvation conditions. (V)acuoles and class (E) compartments. (C) Indicated yeast strains were grown in rich medium (0 hr) or starved for 3 hr. Cell lysates were subjected to SDS-PAGE and western blot analysis with the indicated antibodies. p, precursor form; m, mature form; s, soluble form; *unspecific background band. (D) WT cells, vps4∆ mutants and vps4∆ mutants over-expressing Vps10 were grown to mid-log phase (rich) and starved as indicated. Free amino acids were extracted and analyzed by liquid chromatography. Data are represented as the sum of free amino acids (mg) per gram of dry yeast. Mean ± SD, n = 3. (E) 35S-Met/Cys incorporation into proteins of WT (vps10∆ + VPS10, 2 µ), vps4∆ mutants and vps4∆ mutants over-expressing Vps10 under rich growth conditions and during starvation, was analyzed by SDS-PAGE and digital autoradiography. Coomassie staining shows equal protein loading. (F) Quantification of 35S-incorporation under rich conditions and after 1, 2 and 4 hr of starvation by liquid scintillation counting. Incorporation under rich conditions was set to 100%. Mean ± SEM, n = 3.