Panel A shows confocal fluorescence microscopy images comparing NVJ protein localization in wild-type and ypf1 cells. Panel B shows confocal fluorescence microscopy images comparing Ypf1-GFP and Hmg1-GFP localization in mutant cells. Panel C shows confocal fluorescence microscopy images analyzing catalytically inactive Ypf1 mutant localization patterns. Panel D shows immunoblots comparing protein accumulation under control and glucose-starved conditions. Panel E shows schematic diagram illustrating Ypf1 transmembrane domains, glycosylation, and glutamic acid residues. Panel F shows confocal fluorescence microscopy images comparing localization of Ypf1 truncation mutants during glucose starvation. Panel G shows confocal fluorescence microscopy images examining localization of mutant Nvj1 proteins with Ypf1-GFP. Panel H shows confocal fluorescence microscopy images analyzing Ypf1 and Nvj1 mutant colocalization patterns. Panel I shows schematic model illustrating NVJ remodeling under glucose-starved cellular conditions. Panel J shows confocal fluorescence microscopy images comparing Ypf1-GFP and Hmg1-GFP localization in regulatory mutants. Panel K shows immunoblots analyzing protein accumulation in mutant cells during glucose starvation. Panel L shows confocal fluorescence microscopy images comparing Nvj1-GFP localization between wild-type and snf1 mutant cells.
GS-dependent NVJ partitioning depends on Ypf1 and Snf1. (A) WT and ypf1Δ cells expressing the indicated NVJ proteins fused to GFP or mCherry were observed by confocal fluorescence microscopy after a 24-h GS treatment. (B) WT and nsg1Δnsg2Δ cells expressing Ypf1-GFP or Hmg1-GFP were imaged by confocal fluorescence microscopy after a 24-h GS treatment. (C) A CEN plasmid encoding catalytically inactive Ypf1-D366A/D411A-GFP or -mCherry was introduced into ypf1Δ cells expressing mCherry-Nsg1, GFP-Nsg2, Hmg1-GFP, or Hmg2-GFP, and cells were analyzed by confocal fluorescence microscopy after 1 day of GS. (D) Immunoblotting of whole-cell extracts prepared from WT and ypf1Δ cells grown with or without a 24-h GS treatment. (E) A schematic diagram of Ypf1 showing its transmembrane domains (black boxes). Red lines indicate asparagine residues that undergo N-glycosylation, and blue lines indicate glutamic acid residues. (F–H) Yeast cells expressing C-terminally GFP or mCherry-fused Ypf1 and Nvj1 mutants were imaged by confocal fluorescence microscopy after 1 day of GS. (I) Schematic model of the NVJ remodeling under GS. Color differences in the nuclear ER (nER) indicate predicted changes in membrane properties. (J) The indicated cells expressing the Ypf1-GFP or Hmg1-GFP were imaged by confocal fluorescence microscopy after a 24-h GS treatment. All images shown in A–C, F–H, and J are single focal plane. Scale bar, 5 μm. (K) Immunoblotting of whole-cell extracts prepared from the indicated cells grown with or without a 24-h GS treatment. (L) WT and snf1Δ cells expressing Nvj1-GFP were observed by confocal fluorescence microscopy after a 24-h GS treatment. Maximum projection images reconstituted from z-stacks are shown. Scale bars, 5 µm. Asterisks indicate nonspecific bands. Source data are available for this figure: SourceData F3.