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

To determine which regions of Kre2p are sufficient to target a reporter protein to the Golgi complex, chimeric proteins KKP and KKKP were constructed (see Fig. 6 and Materials and Methods for details). KKP consists of the Kre2p cytoplasmic and membrane-spanning domains fused to the Pho8p luminal domain. KKKP contains the Kre2p NH2 terminus, TMD, and a segment encompassing the first 36 amino acid residues of the stem domain fused to and containing the different chimeric constructions on YEp352 were fixed, spheroplasted, attached to polylysine-coated glass slides, and incubated with anti-Kre2p antibodies which was followed by a subsequent incubation with a FITC-coupled anti-rabbit secondary Ab and DAPI. Colocalization of vacuolar localized chimeric proteins with the yeast vacuolar membrane H÷-ATPase was achieved by coincubation of fixed cells with mAb 13Dll (Kane et al., 1992) with subsequent incubation of treated cells with Texas red-conjugated anti-mouse secondary antibodies.