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

the Pho8p luminal domain. Both chimeric proteins were first verified to be membrane associated and properly glycosylated (data not shown), indicative of a correct type II membrane orientation (Fig. 6) . Chimeric protein KKKP was found, like wild-type Kre2p, to be typically localized to punctiform Golgi structures (Fig. 10) . However, when the partial stem domain was removed, the majority of the resulting chimeric protein (KKP) was found to be localized in the vacuole. The intracellular localization of chimeric protein KKP was quantitatively scored (see Materials and Methods); 70% of positive cells displayed a vacuolar signal and 26% of the cells had vacuolar and punctate fluorescent signals (typical results are shown in Fig. 10) , whereas 4% showed exclusively punctiform fluorescence. Thus, the Kre2 protein region encompassing the NH2 terminus, TMD, and partial stem domain is sufficient for full Golgi retention of a vacuolar protein. These results were extended biochemically by examining the processing kinetics of both chimeric proteins. The PhoSp alkaline phosphatase is activated in the vacuole (Klionsky and Emr, 1989; Nothwehr et al., 1993) where the catalytic luminal domain is processed by the proteinase A (Ammerer et al., 1986; Woolford et al., 1986) . KKKP was found not to be cleaved, whereas in contrast, the majority of KKP was processed in accord with its mostly vacuolar location (data not shown).