Fig. 1.

Expression of E. coli UPPS in S. cerevisiae rer2Δ mutant complements defects in growth and protein N-glycosylation. (A) S. cerevisiae rer2Δ cells were transformed with the empty plasmid YEp352, UPPS, or RER2, under transcriptional regulation of the putative yeast RER2 promoter, plated onto YPD agar and cultured for 3 days at 30°C, as described in “Materials and methods”. (B) Extracts from S. cerevisiae cells were separated by SDS-PAGE (8% acrylamide) and analyzed by Western blot with anti-CPY antibody (Zufferey et al. 1995). The positions of mature CPY (mCPY) and hypoglycosylated glycoforms lacking 1-4 N-linked oligosaccharide chains are indicated (-1 to -4). rer2Δ cells (lane 1) were transformed with YEp352 containing either empty vector (lane 2), UPPS (lane 3), hCIT (lane 4), or RER2 (lane 5), under transcriptional regulation of the putative yeast RER2 promoter, cultured in either YPD or ura selection medium (transformed cell lines).