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

The modification of Kexlp by the Golgi ot(l~3) mannosyltransferase suggested that Kexlp reached the Golgi compartment containing the mannosyltransferase, yet little Kexlpdependent activity was detected at the cell surface (see below) and indicated that Kexlp was retained within the secretory pathway. An analysis of mutant forms of Kexlp was undertaken to examine which domain(s) of Kexlp was responsible for its retention. The hydrophobic domain near the carboxy terminus of Kexlp was previously shown to be responsible for conferring membrane association, as deletion of this 17-amino acid residue region resulted in a soluble protein, Kexlp-AMS (Cooper and Bussey, 1989) . Cells expressing this protein showed a significant increase in Kexlp-dependent activity in the cell medium (see below). That Kexlp-AMS was secreted rather than retained suggests that one of several domains may be responsible for retention of Kexlp: (1) the membranespanning domain which was absent in Kexlp-AMS, (2) the carboxy-terminal domain which was no longer exposed in the cytoplasm in Kexlp-AMS, and (3) a domain amino- S86-16/pKXl-18Hpa was radiolabeled for a 10-min pulse (P) and harvested or chased for an additional 90 min (C). Tunicamycin treatment (TCM) and high pH sodium carbonate extraction were performed as described previously (Cooper and Bussey, 1989) . The forms of Kexlp were immunoprecipitated and analyzed by SDS-PAGE and fluorography.