Rcy1 and v-SNAREs are required for unconventional secretion. (A and B) WT, rcy1Δ, or snc1Δ snc2-V39A,M42A cells were grown in the logarithmic phase, washed twice, and cultured in 2% potassium acetate for 2.5 h. The cell wall proteins were extracted from an equal number of cells followed by precipitation with TCA (“secreted”). Lysates and secreted proteins were analyzed by western blot, and the ratio of the secreted to lysate for the indicated protein was determined and compared with that of WT in each experiment. Statistical analyses were performed for the indicated unconventional cargo proteins, and the reduction in secretion compared with WT is indicated as ± SD. Source data are available for this figure: SourceData F3.