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

Kexlp activity was assayed in whole cells and their growth media to detect any potential mislocalization of the truncated forms of Kexlp to the cell surface. To compare mutants, it was important that the KEX/gene copy number be constant. The Kexlp truncation constructs were, therefore, inserted into the vector YCp50, a centromeric-based plasmid which is normally maintained at a single copy per cell (Rose et al., The indicated strains and transformants were inoculated in either YEPD (A) or YEP + galaetose (B) to a high cell density and grown to stationary phase. The intact cells were washed twice in water and a portion were lysed in 50 mM succinic acid (pH 6.0) with glass beads. The growth media, whole cells, and lysed cells were then assayed for Kexlp activity as described (Cooper and Bussey, 1989) . A unit is defined as 1 pmol of product produced per minute at 30"C. (Error -I-5%.) 1987). Construction of these plasmids resulted in the deletion of an upstream portion of the K/Ds This deletion resulted in reduced production of the encoded proteins relative to wild-type levels. These constructs (the pKX1-20 series of plasmids) were transformed into the yeast strain $86-17 (kex/-A3), a derivative ofS86 in which the KEX/gene had been disrupted. The transformants were grown in liquid selective media; and the media, whole cells, and solubilized lysed cells were assayed for Kexlp activity (data not shown). $86-17 produced no activity and the plasmid-borne wildtype KEX/gene partially restored activity (10% of the level produced by the genomic allele of KEX/) consistent with the promoter truncation.