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

Immunofluorescence Studies fl-Galactosidase-Kexlp fusion protein (Cooper and Bussey, 1989 ) was isolated by preparative SDS-PAGE; electmeluted into 0.I M NI-~CO3, 0.1% SDS; lyophilised; resuspended in coupling buffer (0.5 M NaCl, 0.1 M NaHCO3, pH 9.0); and then dialyzed against coupling buffer + 0.25% SDS + 80 mg ml -~ PMSF. The extract was then coupled to 2 g of activated CNBr-Sepharose 413 (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) overnight at room temperature. The coupling efficiency was judged to be > 80 % as determined by comparing unbound protein with the stming material. The Sepharose was incubated with blocking buffer (1 M ethanolamine, 0.1 M NaHCO3, pH 8.0, 0.5 M NaCI) for 4 h at room temperature. The resulting column (bed volume, 2 ml) was then treated with successive washes of coupling buffer and acetate buffer (0.1 M sodium acetate, pH 4.0, 0.5 M NaC1), and finally with PBS. A similar column (bed volume, 2 ml) was made with an extract made from the granules of E. coli cells producing/~-galactosidase.