Journal: bioRxiv

Article Title: Proteasome-dependent degradation and nucleus–vacuole junctions sustain proteostasis during acute glucose starvation

doi: 10.64898/2026.04.22.720209

Figure Lengend Snippet: A) Representative immunoblots showing turnover of mNeonGreen-tGnd1-HA following 90 min of acute starvation (0.02% glucose) or in glucose-replete control medium (2% glucose). Protein stability was monitored by cycloheximide chase assay (100 µg/mL) at the indicated time points. Total protein loading was visualized and normalized using Stain-Free technology. Graphs represent mNG-tGnd1-HA protein levels as a percentage of the protein present at the time point 0 min. Data are presented as average ± SD from 2 independent experiments. B) Representative confocal fluorescence microscopy images logarithmically growing S. cerevisiae expressing mNeonGreen-tGnd1-HA, which were cultured in glucose-replete (2%) or glucose-deprived (0.02%) media for 90 min, followed by CHX chase (0 and 60 min) prior to fixation and imaging. Images represent maximum intensity projections of Z-stacks. Scale bar, 10 µ m. The bar chart shows the percentage of cells in the culture that contain inclusions, along with the distribution of cells harboring one and two or more inclusions. Data are presented as average ± SD from 2 independent experiments. C) Representative immunoblots showing turnover of mNeonGreen-stGnd1-HA after 90 min acute starvation (0.02% glucose) or control medium (2% glucose), assessed by cycloheximide chase assay (100 µg/mL) at indicated times (0, 60 min). Total protein loading was visualized using Stain-Free technology. Graphs represent mNG-stGnd1-protein levels as a percentage of the protein present at the time point 0 min, with average values and standard deviation (n = 2). D) Representative confocal fluorescence microscopy images of log-phase yeast cells expressing mNeonGreen-stGnd1-HA under glucose-rich YPD (2%) or glucose-deprived (0.02%) conditions for 90 min. Images represent maximum intensity projections of Z-stacks. Scale bar, 10 µ m.

Article Snippet: To investigate the cellular response to acute glucose starvation, yeast Saccharomyces cerevisiae cultures were grown in rich YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose; Formedium Ltd.).

Techniques: Western Blot, Control, Staining, Fluorescence, Microscopy, Expressing, Cell Culture, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Proteasome-dependent degradation and nucleus–vacuole junctions sustain proteostasis during acute glucose starvation

doi: 10.64898/2026.04.22.720209

Figure Lengend Snippet: A) Representative confocal fluorescence microscopy images of log-phase yeast cells expressing HA-mNG-NBD2* under glucose-rich YPD (2%) or glucose-deprived (0.02%) conditions for 90 min. Images represent maximum intensity projections of Z-stacks. Scale bar, 10 µ m. The bar chart depicts the proportion of cells in the culture that contain inclusions. Data are expressed as mean ± SD from two independent experiments. B) Representative immunoblots showing turnover of the model misfolded protein HA-mNG-NBD2*. Log-phase yeast cells were subjected to 90 min acute glucose starvation (0.02% glucose) or control 2% glucose YPD medium, followed by cycloheximide chase assay (100 µ g/mL) at the indicated time points. Total protein loading was visualized using Stain-Free technology.

Article Snippet: To investigate the cellular response to acute glucose starvation, yeast Saccharomyces cerevisiae cultures were grown in rich YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) D-glucose; Formedium Ltd.).

Techniques: Fluorescence, Microscopy, Expressing, Western Blot, Control, Staining

Journal: mBio

Article Title: G521 is the gatekeeper and a key transmembrane domain contact residue of Candida albicans Cdr1

doi: 10.1128/mbio.03746-25

Figure Lengend Snippet: Characterization of crude PMs isolated from logarithmic phase AD1-8u - cells. The following strains were used for these investigations: six hypersusceptible AD1-8u - derivative strains with (AD-pABC3, ADΔ-pABC3, ADΔΔ-pABC3) and without (AD, ADΔ, ADΔΔ) the empty transformation cassette (i.e., with and without the URA3 marker) and ADΔΔ cells overexpressing either the inactive CDR1-E1027Q mutant or wild-type CDR1 each with a C-terminal GFP-His double tag . ( A ) SDS-PAGE of 30 µg crude PM proteins of logarithmic cells of the indicated strains grown in YPD medium, harvested and frozen immediately (left lanes) or starved in ice-cold water for 1 h (lanes to the right of the molecular weight marker) before cell harvest. Arrows indicate the ~200 kDa CaCdr1-GFP-His double band (CaCdr1-GFP-His runs as a double band because most of the C-terminal Gfp tag was not fully denatured even in the presence of 2% SDS and after 5 min denaturation at 50°C), the ~110 kDa Pma1 and the prominent ~50 kDa protein band that was used as a loading control. The % protein expression levels of Cdr1, Pma1, and 50 kDa proteins relative to the bands indicated with * are listed underneath the image. ( B, C ) demonstrate the effects of glucose starvation, the quality (fresh versus frozen), and storage duration (4, 7, and 10 days at −20°C) on ( B ) the Cdr1-specific and ( C ) the Pma1-specific ATPase activities of the six AD1-8u - strains (green bars) and of ADΔΔ cells overexpressing either wild-type CDR1 (red bars) or CDR1-E1027Q (blue bars). Cdr1 = the oligomycin-sensitive (OLI-S) ATPase activity minus the average OLI-S background ATPase activity of the six AD1-8u - control strains; Pma1 = the vanadate-sensitive (VAN-S) ATPase activity minus the OLI-S ATPase activity.

Article Snippet: Yeast cells used for PM preparation were cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose; Difco) at 30°C.

Techniques: Isolation, Transformation Assay, Marker, Mutagenesis, SDS Page, Molecular Weight, Control, Expressing, Activity Assay