Wild type and deletion strains were grown to mid-logarithmic phase, washed twice, and incubated in 2% potassium acetate for 2.5 hr. Cell wall proteins were extracted from equal numbers cells in 100 mM Tris-HCl pH 9.4, 2% sorbitol for 10 min on ice followed by precipitation with TCA. Lysates and cell wall-extracted proteins were analyzed by western blot. The ratio of wall/lysate Acb1 during starvation was determined and compared to that of wild type in each experiment. Statistical analyses were performed for each gene deletion and are represented as% of wild type (paired student’s t-test). (A) Grh1 is required for Acb1 secretion. Representative cell wall extractions monitored by western blot of wild type and grh1Δ cells. (B) grh1Δ cells secreted on average 80.3% less Acb1 than wild-type cells (p = 0.0045, SEM = 3.8%, n= 4). (C) Acb1 secretion requires ESCRT complexes I, II and III but not ESCRT-0. Representative cell wall extractions monitored by western blot of wild type and one deletion strain from each ESCRT complex. (D) Quantification of all ESCRT deletion strains tested (n = 4 or more). ESCRT-0; vps27Δ (+17%, SEM = 3.2%, p > 0.2), hse1Δ (+8%, SEM = 0.9%, p > 0.2). ESCRT-I; vps23Δ (-81.8%, SEM = 7.9%, p = 0.0046), vps37Δ (-81.5%, SEM = 8.4%, p = 0.001), vps28Δ (-69.4%, SEM = 11.8%, p = 0.026). ESCRT-II; vps25Δ (-78.8%, SEM = 14.1%, p = 0.021), vps36Δ (-81.7%, SEM = 2.9%, p = 0.0055), vps22Δ (-71.6%, SEM = 7.7%, p = 0.032). ESCRT-III; snf7Δ (-68.5%, SEM = 4.4%, p = 0.055), vps20Δ (-71.5%, SEM = 6.5%, p = 0.022), vps24Δ (-77.4%, SEM = 7.5%, p = 0.049), vps2Δ (-66.7%, SEM = 11.3%, p = 0.0006). (E) Vps4 and accessory proteins are not required for Acb1 secretion. Representative cell wall extractions monitored by western blot of wild type and indicated deletion strains. (F) Quantification of indicated deletion strains (n = 4 or more). No differences were determined to be statistically significant.