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

The function of the cytoplasmic domain of Kre2p was examined with chimeric protein D K K K which possesses the cytoplasmic NH2 terminus of Kre2p replaced by that of D P A P B. D K K K could not be visualized in SEY6210 (data not shown) suggesting possible degradation of this protein in the yeast vacuole as found with other mistargeted Golgi proteins (Cooper and Bussey, 1992; Roberts et al., 1992; Wilcox et al., 1992; Nothwehr et al., 1993) . To Figure 6 . Schematic representation of chimeric proteins used in the analysis of Kre2p Golgi targeting. The Kre2 protein is composed of 442 amino acid residues. The length of the different Kre2p domains are as follows: NH2 terminus, 11 residues; TMD, 19 residues; stem, ~87 residues and the catalytic domain is ~325 amino acid residues long. DPAP B is an 841-amino acid residue type II vacuolar membrane protein with cytoplasmic NH2-terminal and membrane-spanning domains of 29 and 16 amino acid residues, respectively, and a luminal domain consisting of 796 amino acid residues. The vacuolar alkaline phosphatase Pho8p is a 566-amino acid residue type II membrane protein. The length of the different Pho8p domains are as follows: NHz terminus, 33 residues; TMD, 26 residues; and the luminal domain is 507 amino acid residues long. The different chimeric constructions were made by replacing or fusing specific Kre2p domains with those of vacuolar DPAP B and Pho8p. Chimeric proteins are represented (not to scale) as an assembly of different domains which are distinguished by a letter specifying their origin. K, D, and P, respectively, denote Kre2p, DPAP B, and Pho8p. MR represents the two remaining amino acid residues in the cytoplasmic NH2 terminus of Kre2p after truncation. Putative N-glycosylation sites are indicated (Y). For details on constructions, see Materials and Methods.