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

Immunolocalization results from the different chimeric proteins demonstrate that the Kre2p NH2-terminal cytoplasmic domain was required for localization to the Golgi complex. Chimeric proteins lacking the 11-amino acid residue cytoplasmic domain are fully active as a mannosyltransferase in vitro but fail to be properly retained and are mislocalized to the vacuole. A trivial explanation for the localization of D K K K to the vacuole is that the cytoplasmic domain of DPAP B contains a cryptic vacuolar targeting signal that overrides the Golgi-targeting sequence of Kre2p. Arguing against this view is the demonstration that the NH2-terminal domain of DPAP B is not necessary for vacuolar targeting (Roberts et al., 1992; Nothwehr et al., 1993) . To ensure that mislocalization of DKKK was not caused by some previously unrecognized vacuolar targeting signal of the NH2-terminal domain of DPAP B, chimeric protein MR/KKK was constructed in which the Kre2p cytoplasmic tail was deleted. As with DKKK, MR/ KKK was mislocalized to the vacuole, showing that the NH2-terminal domain of Kre2p is necessary for proper Golgi targeting.