Deletion of ELO3 promotes GS-dependent glycosylation of Ypf1. (A) Glycosylation patterns of Ypf1 were analyzed by immunoblotting using total cell lysates prepared from the knockout strains listed in Table S5. Lysates were obtained from cells harvested either during logarithmic growth (Log) or after 1 day of GS. (B) Immunoblotting of whole-cell extracts prepared from cells carrying either an empty vector or a multi-copy plasmid encoding the ELO3 gene, following GS treatment for the indicated time periods. Line graphs show relative protein levels normalized to the protein abundance of the indicated protein in WT cells at 24 h, which was defined as 100%. For Ypf1, values indicate the proportion of the doubly glycosylated form. Data are means ± SEM (n = 5). Asterisks indicate nonspecific bands. (C) Yeast cells expressing Ypf1-GFP and Nvj1-mCherry and carrying either an empty vector or a 2 µm plasmid encoding ELO3 were observed by confocal fluorescence microscopy after 1 day of GS treatment. Bar graph shows the percentage of cells with Ypf1-GFP localized to the NVJ marked by Nvj1-mCherry. Values are means ± SE (n = 3). At least 30 cells were analyzed per biological replicate. ns: not significant, **P < 0.01. P values were obtained using the unpaired two-tailed t test. Source data are available for this figure: SourceData FS4.