Atg9 is epistatic to Ypt1 in macro-ER-phagy and not to the ER-exit regulator Sec12, which is not defective in this process.

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