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

Yeast total-ceU protein extracts were prepared from cultures growing exponentially in yeast nitrogen base selective medium by cell lysis with glass beads in the presence of protease inhibitors. Membrane fractions were prepared as described (Nakayama et al., 1992) by centrifuging cell lysates at 10,000 g for 20 min and by centrifuging the resulting supernatant at 100,000 g for 1 h. The high speed pellet contained the insoluble membrane fraction. Yeast proteins were separated by SDS-PAGE, and immunoblots were carried out mainly as described (Lussier et al., 1995a) . Briefly, blots were treated in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaC1, 0.05% Tween 20, 5% nonfat dried milk powder) and subsequently incubated with affinity-purified anti-Kre2p antibodies in the same buffer. After antibody binding, membranes were washed in TBST and a second antibody directed against rabbit immunoglobulins and conjugated with alkaline phosphatase, was then added. The blots were again washed and proteins detected using an enhanced chemiluminescence procedure (Amersham Canada, OakviUe, Ontario).