GFP-Snc1-PEM accumulates in APs of <i>vps21∆</i> mutant cells and macro-ER-phagy is independent of UPR induction.

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