10.1371/journal.pgen.1005390.g005 Zhanna Lipatova Zhanna Lipatova Nava Segev Nava Segev Snq2-yEGFP is another macro-ER-phagy cargo and its co-overexpression with DsRed-Snc1-PEM has a synergistic effect. Public Library of Science 2015 erad ER stress degradation ERQC sequential steps core Atgs autophagy use autophagic organelles Ypt GTPases ER membrane upr ap system shuttles misfolded proteins vacuole role pathway ER quality control 2015-07-16 02:50:24 Figure https://plos.figshare.com/articles/figure/_Snq2_yEGFP_is_another_macro_ER_phagy_cargo_and_its_co_overexpression_with_DsRed_Snc1_PEM_has_a_synergistic_effect_/1485808 <p><b>A-C.</b> Overexpressed Snq2-GFP accumulates in the ER and induces UPR in <i>ypt1-1</i> mutant cells. Snq2-yEGFP was overexpressed in WT and <i>ypt1-1</i> mutant cells and the following effects were analyzed as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1A–1C</a>, respectively: increase in the level of Snq2-yEGFP protein (<b>A</b>), accumulation of Snq2-yEGFP in aberrant ER structures (<b>B</b>), and induction of the UPR response (<b>C,</b> GFP-Snc1-PEM is shown for comparison). <b>B.</b> The ER marker Sec61 was tagged with mCherry at its C-terminus in WT and <i>ypt1-1</i> mutant cells. Shown from left to right: DIC, GFP, mCherry, Merge, % cells with intracellular Snq2 and % cells in which Snq2 co-localized with Sec61. <b>D-F.</b> Synergistic effect of co-overexpression of Snq2-GFP with DsRed-Snc1-PEM in <i>atg11∆</i> mutant cells. WT (left) and <i>atg11∆</i> mutant cells (right) were transformed with plasmids for overexpression of Snq2, Snc1-PEM or both Snq2 and Snc1-PEM. <b>D.</b> Immunoblot analysis and quantification. Shown from top to bottom: Snq2-yEGFP (using anti-GFP antibodies), Ds-Red-Snc1-PEM (using anti-Snc1 antibodies), G6PDH (loading control), and a bar graph showing fold increase of Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) in <i>atg11∆</i> mutant cells over WT. <b>E.</b> Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.s005" target="_blank">S5A Fig</a>). <b>F.</b> Fluorescence level of intracellular structures (ratio over PM) in <i>atg11∆</i> mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in <i>atg11∆</i> mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p>