10.1371/journal.pgen.1005390.g004 Zhanna Lipatova Zhanna Lipatova Nava Segev Nava Segev GFP-Snc1-PEM accumulates in APs of <i>vps21∆</i> mutant cells and macro-ER-phagy is independent of UPR induction. 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/_GFP_Snc1_PEM_accumulates_in_APs_of_vps21_8710_mutant_cells_and_macro_ER_phagy_is_independent_of_UPR_induction_/1485807 <p><b>A.</b> The <i>ypt1-1</i> mutation is epistatic to <i>vps21∆</i> in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, <i>vps21∆</i>, <i>ypt1-1</i> and <i>ypt1-1 vps21∆</i> double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of <i>vps21∆</i> mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the <i>vps21∆</i> mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% <i>ypt1-1</i> and <i>ypt1-1 vps21∆</i> mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize. <b>B-D.</b> UPR induction is not required for macro-ER-phagy. The UPR regulators Ire1 or Hac1 were deleted in the WT and <i>ypt1-1</i> mutant cells. The following effects of overexpression of GFP-Snc1-PEM in WT and <i>ypt1-1</i> mutant cells, without and with <i>ire1∆</i> or <i>hac1∆</i>, 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: protein level (<b>B</b>), accumulation of GFP-Snc1-PEM in aberrant structures (<b>C</b>), and UPR induction (<b>D</b>). <b>B.</b> Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or <i>ypt1-1</i> mutant cells. <b>C.</b> Deletion of either Ire1 or Hac1 does not affect the percent of WT and <i>ypt1-1</i> mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. <b>D.</b> Deletion of either Ire1 or Hac1 obliterate UPR in both WT and <i>ypt1-1</i> mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p>