Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane

A different approach to reduce potential degradation of truncated forms of Kexlp was taken where transformants were grown in selective conditions (minimal media) and then transferred to nonselective YEPD media for several generations before assay. YEPD, a medium consisting primarily of yeast extract and peptone, should provide a substrate "buffer" against proteolytic degradation. Although transformants were grown temporarily under nonselective conditions, plasmid loss was never >2% (data not shown). Activity partitioning data suggested that Kexlp-Xho, -AS, and -AMS were secreted from the cell while the other constructs (Kexlp, Kexlp-Hpa, and Kexlp-Hinc) remained intracellular (Table I) . All the mutant forms of Kexlp had approximately the same total activity as the plasmid-borne wild type. The percentage of Kexlp extracellular activity for the membrane-associated forms of Kexlp was comparable to the percentage of cells that stained positively with the vital dye methylene blue, suggesting that such extracellular activity resulted from cell lysis. KEX/was placed downstream from the GAL/ promoter which, upon induction by growth on galactose, resulted in a 70-fold increase over endogenous Kexlp activity. However, such overproduction did not increase the percentage of extracellular Kexlp activity relative to wild-type levels (Table I) .