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

Indirect immunofluorescent detection of Kexlp and Kexlp-Hpa was undertaken to determine if the location of the two proteins differed. The expression of the proteins was such that a Kexlp signal could only be detected upon overproduction of Kexlp (Fig. 7) . Kexlp was found to be localized to a number of small punctate structures (on average, ,x,3-5 per cell) which were not associated with mitochondria, nuclei, or vacuoles, but were characteristic of proteins localized to (Hpa) , or pKX1-20-Hinc (Hinc). Transformants were grown to stationary phase in minimal media, harvested by centrifugation, washed in H20, pelleted, and than resusponded in H20 to a concentration of 5 x 108 cells ml -~. 10/~1 was then placed onto a minimal plate (pH 4.7), seeded with $6 (a strain sensitive to K1 killer toxin). Plates were then incubated at 18"C for 24 h and zones of toxin killing examined. Approximate relative toxin activities, determined by comparison with a toxin dilution series were WT 100%, Xho 30%, AS 40%, AMS 15%, Hpa 55%, and Hinc 55%. (B) Sc25k-13 (kex/), Sc25k (KEX1), and Sc25k-13/pKXI-20WT (WT) were grown and spotted onto a plate seeded with a strain sensitive to K1 killer toxin and treated as described above.