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

To confirm this possibility, colocalization studies were undertaken between the 60-kD subunit of the S. cerevisiae vacuolar membrane H § and either Kexlp or Kexlp-Hpa. The vacuole (as determined by Nomarski optics) correlated to the ring structure in which the 60-kD ATPase subunit was localized by indirect immunofluorescence. The level of Kexlp detected varied among cells due to the variable plasmid copy number (2 #m based). Observation of cells expressing both low and high levels of Kexlp-Hpa (as deter- mined by the intensity of the fluorescence) indicated that >90% of the protein was located in the vacuole. A small percentage of cells (<5 %) showed both ER and vacuolar staining. The protein was associated with the vacuolar membrane demonstrating that it remained membrane associated (Fig. 8) . Kexlp was predominantly restricted to punctate structures indicative of a yeast Golgi location (Fig. 7) . However, 10-15 % of the stained cells expressed high levels of Kexlp (as determined by the intensity of the fluorescence) and showed Kexlp in both Golgi-like structures, and associated with the vacuolar membrane as defined by the 60-kD ATPase subunit (data not shown). No Kexlp or Kexlp-Hpa signal was detected at the plasma membrane. Further indirect immunofluorescence analysis also localized the other membrane-associated forms, Kexlp-Bcl and Kexlp-Hinc, to the vacuolar membrane (data not shown).