10.1371/journal.pgen.1005390.g003 Zhanna Lipatova Zhanna Lipatova Nava Segev Nava Segev Atg9 is epistatic to Ypt1 in macro-ER-phagy and not to the ER-exit regulator Sec12, which is not defective in this process. 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/_Atg9_is_epistatic_to_Ypt1_in_macro_ER_phagy_and_not_to_the_ER_exit_regulator_Sec12_which_is_not_defective_in_this_process_/1485806 <p><b>A-C.</b><i>ATG9</i> was deleted in <i>ypt1-1</i>, WT and <i>sec12ts</i> mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed 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. <b>A.</b> Snc1-PEM is increased ~3 fold in <i>atg9∆</i> and <i>sec12ts</i> as compared to ~20 fold in <i>ypt1-1</i> mutant cells. Importantly, <i>atg9∆</i> is epstatic to the <i>ypt1-1</i>, but not to the <i>sec12ts</i>, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data. <b>B.</b> Whereas deletion of <i>ATG9</i> in <i>ypt1-1</i> mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in <i>sec12ts</i> mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. <b>C.</b> Deletion of <i>ATG9</i> in <i>ypt1-1</i> mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in <i>sec12ts</i> mutant cells (p-value = 0.05). <b>D.</b> Atg9 is present on aberrant ER structures that accumulate in <i>ypt1-1</i> mutant cells. WT and <i>ypt1-1</i> mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the <i>ypt1-1</i> mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed). <b>E.</b> GFP-Snc1-PEM is delivered to the vacuole for degradation in <i>sec12ts</i> mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of <i>sec12ts</i> mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of <i>PEP4</i> in <i>sec12ts</i> mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p>