The general Atgs Atg1 and Atg8 and the selective Atg Atg11, but not other selective Atgs, are required for macro-ER-phagy.

2015-07-16T02:50:24Z (GMT) by Zhanna Lipatova Nava Segev
<p><b>A.</b> GFP-Snc1-PEM protein accumulates in <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells. Lysates were prepared from wild type (WT), <i>ypt1-1</i> (for comparison), <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control. <b>B.</b> GFP-Snc1-PEM protein accumulates in aberrant ER structures in <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61. <b>C.</b> UPR is induced in <i>atg1∆</i>, <i>atg8∆</i>, and <i>atg11∆</i> mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response. <b>D-E.</b> Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (<b>D</b>), intra-cellular accumulation of GFP-Snc1-PEM, (<b>E</b>), and induction of the UPR response (<b>F</b>). Wild type (WT), <i>atg19∆</i>, <i>atg11∆</i>, <i>atg32∆</i>, and <i>atg36∆</i> mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. <b>E.</b> Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.</p>