Journal: Current Issues in Molecular Biology

Article Title: The 50-nm Free Vesicles Visible in Saccharomyces cerevisiae Are Not COPII-Dependent

doi: 10.3390/cimb47050336

Figure Lengend Snippet: Immunogold labeling (10-nm and 15-nm gold) of ERES and Golgi compartments for Sec13, Sec21, Sec22, and GOS1 on ultrathin cryosections. The type of labeling is shown in images. ( A , I – K ) Labeling for Sec22. ( B – D , F ) Sec21 is present on ERES, cis -Golgi, and medial -Golgi. ( C , D ). Serial Tokuyasu ultrathin cryosections. ( E , G , H ) Sec13 is visible on the ER and ERES. ( L ) GOS1-positive 50-nm vesicles. Red arrow shows the 50-nm vesicle. Scale bar: 100 nm.

Article Snippet: Rabbit polyclonal antibody against GOS1 from Saccharomyces cerevisiae was from CUSABIO (7505 Fannin St., Ste 610, Houston, TX, 77054, USA) (Code CSB-PA009677XA01SVG).

Techniques: Labeling

Journal: Current Issues in Molecular Biology

Article Title: The 50-nm Free Vesicles Visible in Saccharomyces cerevisiae Are Not COPII-Dependent

doi: 10.3390/cimb47050336

Figure Lengend Snippet: Alterations of the fine cell structure of S. cerevisiae containing the ts Sec23 mutation after a temperature shift and NEM treatment. ( A – C ) Heating cells at the restricted temperature (37 °C for 10 min) led to ER accumulation. ( D , E ) Accumulation of ERES in these heated cells. The red box and its magnified view show a COPI-coated bud. ( F ) The formation of large ERES and Golgi stacks after returning to the permissive temperature (17 °C) for 5 min. ( G , H ) Three-dimensional model of a Golgi stack formed during recovery composed of two medial cisternae. ( I ) The accumulation of RPs in the cell treated with NEM. The red box shows a secretory granule unable to fuse with the endocytic invagination. ( J – M ) Treatment of cells with NEM induced the accumulation of vesicles of three main size classes: 40 nm, 50 nm, and 65 and more nm. Immunogold labelling of vesicles within the vesicular aggregates for Sso1 and GOS1 after the blockage of membrane fusion with NEM. Markers and size of the gold are indicated on the images. ( J – L ) The 40–nm vesicles were positive for Sso1. ( M ) The 50-nm vesicles were positive for GOS1. ( N ) The 50-nm vesicles were GOS1-positive, whereas the 40-nm and the 65-nm round profiles were negative. ( O ) Double labeling for Sec31 (10-nm gold: black arrow) and Sec21 (15-nm gold). ( P ) COPI-coated bud (arrow) on the Sec31-positive ERES. ( Q ) The 50-nm vesicles (arrow) negative for Sec22 near the ERES positive for Sec22. Scale bars: 1130 nm ( A – C , I ); 350 nm ( D , E ); 175 nm ( F – H ); 70 nm ( M ); 85 nm ( J , L ); 140 nm ( K ); 200 nm ( N ).

Article Snippet: Rabbit polyclonal antibody against GOS1 from Saccharomyces cerevisiae was from CUSABIO (7505 Fannin St., Ste 610, Houston, TX, 77054, USA) (Code CSB-PA009677XA01SVG).

Techniques: Mutagenesis, Membrane, Labeling

Journal: bioRxiv

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1101/2024.11.22.624728

Figure Lengend Snippet: (A) Representative Western blot showing total levels of Ate1-GFP in W303 wild-type strain treated either with or without 5%NaN 3 for 30 minutes. Ate1-GFP expression was induced by the addition of 0.5% galactose and incubation for 3 hours at 30°C. Empty vector serves as a negative control. Alpha tubulin is used for protein loading control. (B) Left panel; representative Western blot showing Ate1-GFP levels in purified mitochondrial fractions, which was treated with proteinase K (5 μg/ml) to remove proteins that are not protected by the mitochondrial membrane. Alpha-tubulin is used as a marker for cytosolic protein contamination, while mitochondrial intermembrane space protein Cmc2 serves as a marker for mitochondrial proteins. Right panel: showing the quantification of mitochondrial Ate1-GFP levels normalized to mitochondrial protein Cmc2. Error bar denotes SD (N=4), p values were calculated by two tailed student t-test.

Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799) anti-yeast-phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 (gift from Dr. Antonio Barrientos) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].

Techniques: Western Blot, Expressing, Incubation, Plasmid Preparation, Negative Control, Control, Purification, Membrane, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1101/2024.11.22.624728

Figure Lengend Snippet: (A) Yeast cells containing Ate1-GFP and Ate1-GFP-Ras2 that are driven by galactose-inducible promoters (pGal) were allowed to express for 6 hours and then briefly treated with oxidative stressor H 2 O 2 for 10 minutes before the microscopic images were taken for the green fluorescence channel. The regular Ate1-GFP forms puncta-like structures similar as shown in , while Ate1-GFP-Ras2 remains localized to the periphery of the cells. (B) Scheme illustrating the principle of the reporter for the arginylation activity inside yeast cells. The N-terminal ubiquitin domain of the reporter protein DD-β15-mCherryFP will be promptly removed by endogenous de-ubiquitination (de-Ub) enzymes in the cell, exposing the penultimate peptide DD-β15, which is derived from the N-terminus of mouse β-actin and is known to be arginylated in vivo [ , ]. The arginylated N-terminus can be recognized with a specific antibody anti-RDD. Antibodies for mCherryFP (mChFP) and GFP can be used to probe the levels of the reporter protein and the GFP-fused ATE1, respectively. (C) To test the arginylation activity of different forms of Ate1 (Ate1-GFP or Ate1-GFP-Ras2), they were expressed in ate1 Δ yeast (to avoid the interference of endogenous ATE1), which was also simultaneously expressing the reporter protein DD-β15-mCherryFP. The arginylation level of the reporter protein was measured as described in (B). Pgk1 serves as loading controls for the yeast proteins. (D) Growth test of ate1 Δ yeast cells carrying either the empty expression vector or pGAL1:Ate1-GFP and pGAL1:Ate1-GFP-Ras2 was conducted by a serial dilution growth assay on either plate containing glucose or galactose, where the expression of Ate1 is not induced or induced, respectively. (E) Representative Western blots showing the expression levels and distributions of different Ate1 constructs (Ate1-GFP and the mitochondrial matrix-targeting Mt-Ate1-GFP) in total cell lysate (left panel) and in purified mitochondrial fractions (right panel). Vector alone served as a negative control. The expression of both the Ate1 construts was achieved by adding 0.5% galactose and incubated for 3hrs at 30 o C. The expressions and subcellular distributions Ate1-GFP and Mt-Ate1-GFP were probed by anti-GFP. The level of Pgk11 and Cmc2 were used as loading controls for total proteins or mitochondrial proteins, respectively. (F) Growth test of ate1 Δ yeast cells carrying either the empty expression vector, pGAL1:Ate1-GFP, or the mitochondria matrix localized Ate1 (pGAL1: Mt-Ate1-GFP) by a serial dilution growth assay on either plate containing galactose or glucose for the induced expression (or not) of Ate1. Note that the concentration of galactose (0.5%) is lower than elsewhere to allow the display of the difference between the Ate1-GFP and mt-Ate1-GFP. Plates were incubated at 30°C and images were taken after 3 days.

Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799) anti-yeast-phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 (gift from Dr. Antonio Barrientos) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].

Techniques: Fluorescence, Activity Assay, Derivative Assay, In Vivo, Expressing, Plasmid Preparation, Serial Dilution, Growth Assay, Western Blot, Construct, Purification, Negative Control, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1101/2024.11.22.624728

Figure Lengend Snippet: A) Top panel shows the representative Western blot for the level of Grp78/HDEL, a maker of the endoplasmic reticulum stress response, in yeast cells carrying the pYES2-pGAL: ATE1-6xHis- URA3 expression vector or the empty control vector, which were induced with 2% galactose for 6h in liquid media. Pgk1 is used as loading controls. Ate1 was probed with anti-GFP. Bottom panel is a graph indicating the fold differences in GRP78 levels between the Ate1 overexpression (OE) and vector control. Error bar denotes SEM (N=3). B) Top panel shows representative Western blot for the level of full-length Atg8-GFP and the derivative GFP, which is resulted form proteolysis in autophagy. The yeast cells carrying the pYES2-pGAL: ATE1-6xHis- URA3 expression vector or the empty control vector were induced with 2% galactose for 6h in liquid media. Pgk1 is a loading control. Bottom panel is a graph indicating the fold differences in the ratio between GFP and the full-length Atg8-GFP, which reflects the activity of autophagy, in the presence of Ate1 overexpression (OE) and vector control. Error bar denotes SEM (N=3).

Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799) anti-yeast-phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 (gift from Dr. Antonio Barrientos) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Over Expression, Activity Assay

Journal: bioRxiv

Article Title: The C-terminal domain of Hsp70 is responsible for paralog-specific regulation of ribonucleotide reductase

doi: 10.1101/2022.02.08.479504

Figure Lengend Snippet: (A-C) Cells expressing either Ssa1, 2, 3 or 4 as their sole Ssa and HA-tagged Rnr1/Rnr2/Rnr4 were grown to exponential phase and were either left untreated or were treated with 200 mM HU for 3 hrs. HA-RNR complexes were immunoprecipitated with anti-HA magnetic beads and were subjected to SDS-PAGE and analyzed by immunoblotting with anti-HA antibodies to detect the RNR subunits or anti-Ydj1 antibodies to detect Ydj1. (D) Interaction between RNR and Ydj1 (Ydj1/RNR) was calculated by quantitating bands from three replicate experiment. Each value represents the mean ± SD (n = 3). Statistical significance between samples was calculated ANOVA. (∗∗∗∗ p < 0.01).

Article Snippet: Proteins were detected using the following antibodies; anti-HA tag (Thermo #26183), Anti-FLAG tag (Sigma, #F1365), anti-PGK1 (Thermo # PA5-28612), anti-Ydj1 (StressMarq #SMC-166D).

Techniques: Expressing, Immunoprecipitation, Magnetic Beads, SDS Page, Western Blot