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

For dual-labeling experiments involving Kre2p and medialor late Golgi markers, the influenza hemagglutinin virus epitope (sequence YPY-DVPDYA) was inserted by oligonucleotide-directed mutagenesis directly at the COOH-terminal domain of Mnnlp and in the region corresponding to the Kexlp luminal domain. Epitope-tagged Mnnlp and Kexlp were detected with the 12CA5 monoclonal antibody (Kolodziej and Young, 1991) . The latter was used at dilutions ranging from 1:250--1:1,000. mAb 13Dll which recognizes the 60-kD subunit of the yeast vacuolar membrane H+-ATPase (Kane et al., 1992) was used at dilutions of 1:10-1:25 as a vacuolar marker for colocalization studies with Kre2p chimeric proteins. Fluorescence signals were obtained by subsequent incubation of treated cells with rhodamine X sulfonyl chloride (Texas red)-conjugated goat anti-rabbit IgG (1:50-1:200) and FfTC-conjugated goat anti-mouse IgG (1:50-1:200) which were used as secondary antibodies. Nuclei and mitochondria were visualized by staining with 4',6-diamidino-2-phenyl-indole (DAPI). Cells were examined with an epifluorescence microscope (Axiophot; Carl Zeiss, Inc., Thornwood, NY), and photographed with film (T-Max 400; Eastman Kodak Co., Rochester, NY).