General secretion assay

Cells were grown overnight at 25°C to log phase, and 7.5 OD₆₀₀ units were resuspended in 2 ml of fresh methionine-free medium containing 0.06 mg/ml bovine serum albumin and then shifted to 15°C for 60 min. After the incubation, the cells were labeled with 500 μCi of [³⁵S]ProMix at 15°C, and aliquots of cells (350 μl) were removed at the end of the indicated time points. Labeled cell suspensions were centrifuged for 1 min at 13,000 × g, and then 300 μl of the medium (extracellular fractions) was transferred to an ice-cold tube containing 30 μl of stop mix (500 mM NaN₃, 500 mM NaF). The extracellular fractions were centrifuged again for 1 min at 13,000 × g to remove residual cells, and the supernatant was transferred to a fresh tube containing 20 μl of 100% trichloroacetic acid (TCA) and 1 mg/ml sodium deoxycholate. All samples were incubated on ice for 60 min before the TCA precipitates were pelleted at 13,000 × g for 5 min and washed twice with ice-cold acetone. The acetone-washed pellets were air-dried, resuspended in 40 μl of 1X SDS sample buffer, heated to 100°C for 5 min, and subjected to SDS–PAGE using a 6% gel, followed by autoradiography.