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

Initially, the role of the membrane-spanning domain of Kre2p was assessed with fusion protein KDKK (Fig. 6) , in which the Kre2p TMD was removed and substituted with that of DPAP B. KDKK was found, like wild-type Kre2p, to be localized to small punctate structures of the yeast Golgi (Fig. 7) . To exclude the possibility that the DPAP B TMD is able to function as a Kre2p TMD in the context of chimeric protein KDKK by sharing some unknown common features not apparent at the amino acid level, a chimeric protein consisting of Kre2p with the Pho8p membrane-spanning domain was made. As was the case with KDKK, KPKK was also found to be localized to the Golgi Hill et al., 1986) and the influenza hemagglutinin virus epitope-tagged MNN1 on multicopy plasmid YEp352 (URA3; Hill et al., 1986) or epitope-tagged KEX1 gene (see Materials and Methods) under an ADH1 gene promoter on centromeric-based plasmid pRS316 (URA3; Sikorski and Hieter, 1989) were fixed, spheroplasted, attached to polylysine-coated glass slides, and then incubated with affinity-purified anti-Kre2p and 12CA5 mAb (Kolodziej and Young, 1991) . Fluorescence signals were obtained by subsequent incubation of treated cells with Texas red-conjugated goat anti-rabbit IgG and FITC-conjugated goat antimouse IgG which were used as secondary antibodies. Arrows indicate colocalization of punctiform fluorescence. apparatus (Fig. 7) indicating that the Kre2p TMD is dispensable for Golgi retention.