Figure 6. Limitations of the in vivo fusion assay employed by Coonrod et al.

(A) The figure illustrates the mating procedure and the potential fate of proteins expressed from plasmids carrying the PHO8 gene (producing pro-ALP) or the PEP4 gene (producing its maturase proteinase (A) under control of the inducible CUP1 promotor. Induced pro-ALP is indicated by grey circles, pro-ALP resulting from constitutive, non-induced background expression is indicated by green circles. Induced Pep4 is indicated by grey scissors, Pep4 resulting from non-induced background expression by black scissors. For discussion, see main text. (B) Dilution of induced pro-ALP over time. KEBY136 cells²⁵ expressing PHO8 from the CUP1 promotor were grown on SC-URA plus 50 μM CuCl₂ for 24 h and then shifted to YPD without copper. Aliquots of cells were withdrawn before and 24 and 48 h after the shift and analyzed by SDS-PAGE and Western blotting against ALP and actin. Samples for background controls were taken from cells that had never been induced by copper and from pho8Δ cells that lacked the PHO8-expressing plasmid (KEBY136). (C) Conservative estimation of the abundance of induced pro-ALP relative to pro-ALP resulting from non-induced expression of PHO8 from the CUP1 promotor, resulting from the published mating assay for vacuole fusion²⁵. The first 24h period represents a period of cultivation of the cells used to chase pro-ALP from the biosynthetic pathway. The second 24 h culture period that follows mating is used to select for diploid offspring.