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

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posted on 16.07.2015, 02:50 by Zhanna Lipatova, Nava Segev

A. GFP-Snc1-PEM protein accumulates in atg1∆, atg8∆, and atg11∆ mutant cells. Lysates were prepared from wild type (WT), ypt1-1 (for comparison), atg1∆, atg8∆, and atg11∆ 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. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1∆, atg8∆, and atg11∆ 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. C. UPR is induced in atg1∆, atg8∆, and atg11∆ 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. D-E. 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 (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19∆, atg11∆, atg32∆, and atg36∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. 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.