Panel A shows fluorescence images revealing NVJ accumulation during glucose starvation conditions. Panel B shows fluorescence images comparing protein localization in wild-type and nvj1 cells. Panel C shows fluorescence images of Nvj1-GFP localization across multiple mutant strains. Panel D shows immunoblots comparing protein accumulation between wild-type and nvj1 mutant cells. Panel E shows immunoblots and graph tracking protein accumulation during glucose starvation periods. Panel F shows immunoblots analyzing Ypf1 glycosylation changes under glucose starvation conditions. Panel G shows immunoblots examining glycosylation-site mutant effects on Ypf1 modification patterns. Panel H shows immunoblots comparing protein abundance across multiple mutant yeast strains.
Ypf1, Nsg1, and Nsg2 accumulate at the NVJ under glucose-starved conditions. (A) Yeast cells co-expressing Ypf1-GFP/Nvj1-mCherry, mCherry-Nsg1/Nvj1-GFP, or GFP-Nsg2/Nvj1-mCherry were observed by confocal microscopy under the indicated culture conditions. Log, NS, and GS refer to cells grown in logarithmic phase in YPD or subjected to nitrogen or GS for 24 h, respectively. (B) WT and nvj1Δ cells expressing Ypf1-GFP, mCherry-Nsg1, or GFP-Nsg2 were imaged by confocal fluorescence microscopy after a 24-h incubation in GS medium. (C) The indicated yeast cells expressing Nvj1-GFP were observed by confocal fluorescence microscopy after a 24-h incubation in GS medium. (D) Total cell lysates from WT and nvj1Δ cells after a 24-h incubation in GS medium were analyzed by immunoblotting using the indicated antibodies. (E) Total cell lysates were prepared from WT cells grown in GS medium for the indicated periods of time and analyzed by immunoblotting. Line graph shows protein levels relative to those in cells without GS treatment. Values are means ± SE (n = 3). (F and G) Glycosylation patterns of Ypf1-FLAG and its mutants were analyzed by immunoblotting using whole-cell lysates prepared from cells with or without a 2-day GS treatment. (H) Total cell lysates from the cells shown in C were analyzed by immunoblotting. All images shown in A–C are single focal plane. Scale bar, 5 μm. Asterisks indicate nonspecific bands. Source data are available for this figure: SourceData F2.