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

All yeast constructions used strains SEY6210 (MATa, (Boone et al., 1990) . Strains were transformed using the lithium acetate procedure using sheared, denatured carrier DNA (Gietz et al., 1995) . Transformants were selected on synthetic minimal medium with auxotrophic supplements. Levels of sensitivity to K1 killer toxin were evaluated in SEY6210 by a seeded plate assay using a modified medium consisting of 0.67% YNB, 0.0025% required amino acids, 1.0% bacto agar, 0.001% methylene blue, 2% glucose, and buffered to pH 4.7 with Halvorson minimal medium (Lussier et al., 1993 (Lussier et al., , 1995a Brown et al., 1994) . (underlined) . Finally, KPKP was obtained by replacing the Kre2p TMD in KKKP with that of Pho8p using the same oligonucleotide that was used to obtain KPKK All chimeric protein constructions were verified by DNA sequencing using the dideoxy chain termination procedure (Sanger et al., 1977) with the Sequenase enzyme (United States Biochemical Corp., Cleveland, OH), a-asS-dATP and specific DNA primers.