Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to a medial-Golgi compartment

To examine the basis of localization of a yeast Golgi glycosyltransferase, an analysis of Kre2p noncatalytic domains was made. The roles of the cytoplasmic NH2 terminus, TMD, and luminal stem region of Kre2p in Golgi targeting were tested by constructing chimeric proteins in which Kre2p-specific segments were substituted with the corresponding domains of the DAP2 or PH08 gene products (Fig. 6) . DAP2 encodes the vacuolar dipeptidyl aminopeptidase B (Roberts et al., 1989) , DPAP B, and is a type II integral membrane glycoprotein that lacks apparent vacuolar targeting information. No individual domain of DPAP B was shown to be required for its transport to the vacuole besides a nonspecific hydrophobic TMD (Roberts et al., 1992) . PH08 encodes a vacuolar alkaline phosphatase. It is also a type II membrane protein (Kaneko et al., 1987; Klionsky and Emr, 1989; Nothwehr et al., 1993) that is thought to be transported to the vacuole by default (Nothwehr et al., 1993) . Substitutions designed with vacuolar proteins were used to avoid potential problems with cryptic Golgi-targeting sequences. Indirect immunofluorescence detection of chimeric proteins was undertaken to determine their intracellular location(s).