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

Yeast cells were grown in selective medium to an OD600 of 0.5-0.7. and immediately fixed by the addition to cultures of potassium-phosphate (pH 6.5) to 0.1 M, and formaldehyde to 3.7%. After gentle agitation for 30 rain, cells were pelleted and resuspended in a fixation buffer containing 0.1 M potassium-phosphate (pH 6.5) and 4.5% formaldehyde. Cells were further fixed for another 30 rain. Formaldehyde-treated ceils were washed with 0.1 M potassium-phosphate (pH 6.5) buffer, resuspended in a solution of IOO mM Tris-HC1, pH 8, 25 mM DTr, 5 mM EDTA, and 1.2 M sorbitol and incubated for 10 min at 30°C with gentle agitation. After washing of the fixed cells, cell walls were removed by treatment with Zymolyase 100T (ICN Biochemicals) at a final concentration of 200 ixg/ml in 0.1 M potassium phosphate (pH 6.5) 1.2 M sorbitol for 20-30 min at 30°C. Fixed spheroplasted cells were washed in 0.1 M potassium-phosphate (pH 7.4) 1.2 M sorbitol and resuspended in the same buffer. Cells were subsequently adsorbed to poly-L-lysine coated microscope slides, permitted to stand for 10 min and washed with PBS. Slides were then immersed in -20°C methanol for 1-6 min, and then for 20450 s in -20°C acetone, depending on yeast strains. Treated slides were air-dried and used immediately or stored at 4°C until needed. Fixed mounted cells were incubated with primary antibodies diluted in PBS containing 0.5 mg/ml BSA for 1 h at 25°C or overnight at 4°C. Anti-Kre2p Ab was used at dilutions of 1:25-1:100.