Fig 1. General autophagy is not induced during constitutive ER-phagy of an overexpressed membrane protein.

A-C. Overexpression or intracellular accumulation of GFP-Snc1-PEM does not result in elevation of Atg8 protein level. A. atg11Δ mutant cells accumulate 3.5-fold more GFP-Snc1-PEM than WT cells during normal growth. WT and atg11Δ mutant cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM (or empty plasmid as a negative control). Cells were grown either in SD+N medium (left), or in medium without N (right) for 6 hours. The level of GFP-Snc1-PEM in cell lysates was determined using anti-GFP antibodies and immuno-blot analysis (G6PDH was used as a loading control). Shown from top to bottom: growth medium, strain, plasmid, GFP blot, G6PDH blot, and quantification of the GFP-Snc1-PEM band: fold over wt, +/- STD, p-value. B. atg11Δ mutant cells accumulate intracellular GFP-Snc1-PEM during normal growth. Cells from panel A were visualized by live-cell fluorescence microscopy, except that FM4-64 was added to cells during nitrogen starvation. Shown from left to right: growth condition, strain, DIC (for cell contour), GFP, and % cells in which intracellular GFP-Snc1-PEM accumulates in the cytoplasm (not in vacuole); +/-, STD, p-value. GFP-Snc1-PEM and GFP accumulates in the cytoplasm of the majority of atg11Δ mutant cells during normal growth (top), and ~2-fold less under starvation (bottom); (p-value <0.01). Under nitrogen starvation (-N), GFP-Snc1-PEM accumulates in the vacuoles (outlined with FM4-64) of both WT and mutant cells. >100 cells were visualized per panel; arrows and arrowheads point to GFP-Snc1-PEM in the cytoplasm and the vacuole, respectively; size bar, 1μ. C. The level of Atg8 is not increased due to overexpression of GFP-Snc1-PEM during normal growth, but is increased by 5-7-fold during N starvation regardless of GFP-Snc1-PEM overexpression. Cell lysates from panel A were tested by immuno-blot analysis using anti-Atg8 antibodies. Shown from top to bottom: Atg8 blot, G6PDH blot (loading control), quantification of fold Atg8 compared to the level in WT cells (+/-, STD), and p-values >0.05 showing no significant difference (N.S.) with and without Snc1 overexpression. D. Ape1 processing is not induced in atg11Δ mutant cells overexpressing GFP-Snc1-PEM. Cell lysates from panel A were analyzed by immuno-blot analysis for processing of prApe1 to mApe1 using anti-Ape1 antibodies. In WT cells, Ape1 is mostly processed during normal growth and fully processed under stress (-N). In atg11Δ mutant cells, Ape1 is partially processed under stress (-N), but not during normal growth regardless if the cells express GFP-Snc1-PEM. E. Pho8Δ60 phosphatase activity is not induced in WT or atg11Δ mutant cells expressing GFP-Snc1-PEM during normal growth. Pho8Δ60 phosphatase activity was measured in lysates of cells from panel A grown in SD+N (while bars); the activity is elevated in all cells under stress (gray bars, SD-N) regardless if they express GFP-Snc1-PEM. Error bars, STD. Results in this figure represent 3 independent experiments.