Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane

$86-16 transformants were grown to mid-log, and 6 • l0 s cells harvested, washed in I"I20, and resuspended in 3 ml of ZSM (1.2 M sorbitol, 10 mM CaC12, 10 mM Tris-HC1, pH 7.5). 300/~g of Zymolyase 60,000 (ICN Biomedicals, Inc., Costa Mesa, CA) and/~-mercaptcethanol (final concentration 0.4%) were added to the cells before incubation at 30oc for 75 min. The resulting spheroplasts were gently pelleted, washed twice in 1.2 M sorbitol, 10 mM Tris-HC1 (pH Z5), and resuspended in 25 ml of YNB-Sorb (Yeast Nitrogen Base, 2% glucose, 1.2 M sorbitol), and incubated at 30oc for 3 h. Spheroplasts were then concentrated to 5 ml, and BSA (200 mg ml -l) and PMSF (Boehringer Mannheim Corp., Indianapolis, IN; final concentration I raM) were added before the addition of 500 mCi of Tram Label (ICN Biomedicals, Inc.). After 10 rain, half of the cells were harvested and the remainder chased for 30 rain as described above. After labeling, the spheroplasts were placed on ice, and sodium azide (Fisher Scientific Co., Pittsburgh, PA) was added to 1 raM. Spheroplasts were then gently pelleted and both the supernatant and pellet fractions retained. The supernatant was microfuged for 2 rain and the resulting supernatant was concentrated with Centricon 30 (Amicon, Beverly, MA) to a final volume of 200 ~tl before heating to 100~ in the presence of SDS (1%). The spheroplasts werv washed once in ice cold 1.2 M sorbitol, I0 mM Tris-HC1 (pH 7.5), pelleted, resuspended in BSB + 1% SDS (Cooper and Bussey, 1989) , and boiled. The detergent-solubilized samples were then treated as above and immum~recipitated with Kexlp antiserum.