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. 1997 May 5;137(3):609–618. doi: 10.1083/jcb.137.3.609

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Membrane-associated API is imported into the vacuole as an oligomer. The K12R API mutant was pulse labeled for 10 min and chased for 30 min at 38°C and then shifted to 30°C. Samples were removed at the indicated times during the 20–40-min window of the 30°C shift period and lysed with glass beads, and the resulting extract was separated by 20–50% glycerol gradients. Fractions were collected, immunoprecipitated with antiserum to API, and resolved by SDS-PAGE. Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating with the thyroglobulin molecular mass standard (669 kD).