Depletion of Atg9 and Atg2, but not Atg18, result in a macro-ER-phagy phenotype different from that of other core Atgs, and <i>atg9∆</i> is epistatic to <i>atg11∆</i>.

2015-07-16T02:50:24Z (GMT) by Zhanna Lipatova Nava Segev
<p><b>A-C</b>. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), <i>atg9∆</i>, <i>atg18∆</i>, and <i>atg2∆</i> mutant cells overexpressing GFP-Snc1-PEM 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. The tested phenotypes: the level of GFP-Snc1-PEM protein (<b>A</b>), accumulation of GFP-Snc1-PEM in aberrant structures (<b>B</b>), and induction of the UPR response (<b>C,</b><i>atg1∆</i> is shown as a positive control). <b>B.</b> Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. <b>D-G.</b> Atg9 is epistatic to Atg11 in macro-ER-phagy. <i>ATG9</i> was deleted in wild type and <i>atg11∆</i> mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005390#pgen.1005390.g001" target="_blank">Fig 1</a> legend. <b>D.</b> Deletion of <i>ATG9</i> in wild type (WT) or <i>atg11∆</i> mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in <i>atg11∆</i> mutant cells. <b>E.</b> Deletion of <i>ATG9</i> in wild type or <i>atg11∆</i> mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the <i>atg9∆</i> single-, and <i>atg9∆ atg11∆</i> double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of <i>atg11∆</i> mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain). <b>F.</b> UPR is induced in <i>atg11∆</i>, but not in <i>atg9∆</i> single- and <i>atg9∆ atg11∆</i> double-mutant cells overexpressing GFP-Snc1-PEM. <b>G.</b> UPR can be induced in <i>atg9∆</i> single-, and <i>atg9 atg11∆</i> double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p>