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

txl,2-mannosyltransferase activity assays were performed essentially as described (Lewis and Ballou, 1991; Hhusler and Robbins, 1992) . S. cerevisiae cells ($86 background) were grown in selective medium to an OD600 of 0.8-0.9 and lysed with glass beads in the presence of protease inhibitors. High speed pellet fractions including Golgi and vacuolar membranes were prepared by centrifuging cell lysates at 1,1300 g for 20 min and by centrifuging the resulting supernatant at 100,000 g for 1 h at 4°C. Incubation mixtures contained 50 mM Hepes, pH 7.2, 10 mM MnCI2, 0.1% Triton X-100, 0.2 mM GDP-[lgC]mannose, and 10 mM C~-D-methylmanno-pyranoside as an acceptor (Sigma Chemical Co.) and 10 p,1 of membrane fraction in a total vol of 20 tzl. Reaction mixtures were incubated for 30--60 min at 30°C and then passed through a resin (AG1-X4; Bio-Rad Laboratories, Hercules, CA) column to remove unreacted GDP-mannose. Neutral products were eluted with 1 ml of water and radioactivity was measured. Control assays were conducted in which the saccharide acceptor was omitted and counts obtained in these assays were subtracted from values obtained in assays made with the sugar acceptor. Enzymatic activities (see Fig. 9 ) are expressed as percentages of specific activity (mmol/h/mg of membrane protein) for each chimeric protein.