Endosomal function of wild-type, vps4Δ, and vta1Δ cells was assessed by analyzing secretion of the vacuolar hydrolase CPY. The indicated strains were metabolically labeled with [³⁵S]cysteine/methionine and chased with excess cold cysteine/methionine for 30 min, and intracellular and extracellular fractions were immunoprecipitated with anti-CPY antibody and subjected to SDS-PAGE. Immunoreactive material from each fraction was quantitated using the ABI Storm 840 system and quantitated using ABIÕs ImageQuant software package. Conversion of the MVB cargo CPS from its precursor to mature form was also measured as a qualitative measure of MVB sorting. The indicated strains were metabolically labeled with [³⁵S]cysteine/methionine and chased with excess cold cysteine/methionine for 10 min, and time points were taken up to 50 min after addition of chase. Anti-CPS immunoprecipitation reactions were performed on each time point, and immunoreactive material was subjected to SDS-PAGE and quantitation as with CPY.