Panel A: A set of microscopy images showing the localization of various GFP-tagged proteins in wild-type (WT) and ypf1 delta yeast cells. The images are arranged in pairs, with WT on the left and ypf1 delta on the right. Each row represents a different protein, including Nvj1-GFP, Nvj2-GFP, Vac8-GFP, Osh1-GFP, Tsc13-GFP, mCh-Nsg1, GFP-Nsg2, Hmg1-GFP, and Hmg2-GFP. The scale bar is 20 micrometers. Panel B: Confocal fluorescence microscopy images of ypf1 cells expressing either Ypf1-GFP or Ypf1-3NQ-GFP after 2 days of GS treatment. The images show GFP and merged channels. The scale bar is 5 micrometers. Panel C: Confocal fluorescence microscopy images of wild-type and ypf1 cells expressing Hmg1-GFP and harboring either an empty vector, Ypf1-FLAG, or Ypf1-3NQ-FLAG-expressing plasmid after 2 days of GS treatment. The images show DIC and GFP channels, with an enhanced view of the GFP channel. The scale bar is 5 micrometers. Panel D: An immunoblotting analysis of total cell lysates from the indicated strains after 1 day of GS treatment. The blot shows bands corresponding to GFP, Nvj1, Ypf1, Nsg1, Nsg2, and Tim23 proteins.
The glycosylation of Ypf1 is not required for GS-dependent remodeling of the NVJ. (A) Uncropped microscopy images corresponding to those shown in Fig. 3 A. Maximum projection images reconstituted from z-stacks are shown. Scale bar, 20 μm. (B)ypf1Δ cells harboring a CEN plasmid expressing either Ypf1-GFP or the glycosylation-deficient mutant Ypf1-3NQ-GFP were observed by confocal fluorescence microscopy after 2 days of GS. (C) WT and ypf1Δ cells expressing Hmg1-GFP and harboring either an empty vector (−), Ypf1-FLAG, or Ypf1-3NQ-FLAG-expressing plasmid were observed by confocal fluorescence microscopy after 2 days of GS. (D) Total cell lysates from the indicated strains after 1 day of GS were analyzed by immunoblotting. All images in B and C are single focal plane. Scale bar, 5 μm. Source data are available for this figure: SourceData FS3.