Snq2-yEGFP is another macro-ER-phagy cargo and its co-overexpression with DsRed-Snc1-PEM has a synergistic effect.

2015-07-16T02:50:24Z (GMT) by Zhanna Lipatova Nava Segev
<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>