Journal: PLOS Genetics

Article Title: Ubp2 modulates DJ-1-mediated redox-dependent mitochondrial dynamics in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1011353

Figure Lengend Snippet: (A, B) Evaluation of the relative steady-state levels of Fzo1 by western blotting. The cells from indicated strains lysed, and Fzo1 levels were probed in the whole cell lysate (A) . The blots were subjected to quantification by densitometry, and the fold change in Fzo1 expression was represented. (C, D) Assessment of the turnover kinetics of Fzo1 using cycloheximide assay. The cells were subjected to treatment with cycloheximide for the indicated time points, and the lysates were subjected to western blotting (C) . The blots were subjected to quantification densitometrically and represented the rate at which Fzo1 is degraded across the strains (D). Two-way ANOVA was performed using three biological replicates. (E, F) Evaluation of ubiquitination status of Fzo1. Genomically tagged Fzo1 at the C-terminus with HA-tag was subjected to pull-down analysis using HA-conjugated beads and probed with anti-HA and anti-ubiquitin specific antibodies (E) . The Western blots were subjected to quantitation by densitometry, and fold change in the ubiquitination levels is represented (F). One-way ANOVA with Tukey’s multiple comparison tests was used for significance analysis. Error bars represent the standard deviation in median values from 3 biological replicates. Asterisks indicate the p-value, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The blots were incubated with either with anti-HA (Monoclonal anti-HA tag antibody, Catalogue no: SAB2702217, Sigma Aldrich) and anti-ubiquitin antibody (Ubiquitin antibody, Catalogue no: NB300–130, Novus Biologicals).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Quantitation Assay, Comparison, Standard Deviation

Journal: PLOS Genetics

Article Title: Ubp2 modulates DJ-1-mediated redox-dependent mitochondrial dynamics in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1011353

Figure Lengend Snippet: (1) In healthy cells (left panel), DJ-1 homologs maintain optimum redox homeostasis by regulating balanced fusion and fission events. (2) The fusion-fission balance is crucial for maintaining healthy mitochondrial mass and function. (3) The ubiquitination status of Fzo1 dictates either fusion or degradation. Ubp2 mediates fusion by editing the ubiquitin chain on Fzo1, promoting mitochondrial fusion. Ubp12 opposes the function of Ubp2 by antagonizing fusion. (4) In cells lacking DJ-1 paralogs (right panel), the redox status is perturbed due to increased basal ROS levels. (5) The elevated ROS is due to an increase in the functional hyperfused mitochondrial structures, which in turn is used by the cells as an adaptive strategy. (6) The imbalance in fission-fusion dynamics in cells lacking DJ-1 paralogs is attributed to increased Fzo1 levels. The ubiquitination status of Fzo1 is altered due to increased expression of Ubp2, leading to enhanced fusion events and hence perturbation in the mitochondrial dynamics. The model was made using modified mitochondria icons from https://bioicons.com/ (simple_mitochondria_network icon by Marnie-Maddock https://github.com/MarnieMaddock is licensed under CC-BY 4.0 Unported https://creativecommons.org/licenses/by/4.0/ );(mitochondria-grey icon by DBCLS https://togotv.dbcls.jp/en/pics.html is licensed under CC-BY 4.0 Unported https://creativecommons.org/licenses/by/4.0/ ), (mitochondrium-yellow icon by Servier https://smart.servier.com/ is licensed under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/ ) and the rest of the figure was drawn.

Article Snippet: The blots were incubated with either with anti-HA (Monoclonal anti-HA tag antibody, Catalogue no: SAB2702217, Sigma Aldrich) and anti-ubiquitin antibody (Ubiquitin antibody, Catalogue no: NB300–130, Novus Biologicals).

Techniques: Ubiquitin Proteomics, Functional Assay, Expressing, Modification