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. 2014 Sep 15;127(Pt 18):4078-88.
doi: 10.1242/jcs.154716. Epub 2014 Jul 22.

ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery

Sebastian Schuck  1 , Ciara M Gallagher  2 , Peter Walter  2
Affiliations

ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery

Sebastian Schuck et al. J Cell Sci. .

Abstract

Selective autophagy of damaged or redundant organelles is an important mechanism for maintaining cell homeostasis. We found previously that endoplasmic reticulum (ER) stress in the yeast Saccharomyces cerevisiae causes massive ER expansion and triggers the formation of large ER whorls. Here, we show that stress-induced ER whorls are selectively taken up into the vacuole, the yeast lysosome, by a process termed ER-phagy. Import into the vacuole does not involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that ER-phagy is topologically equivalent to microautophagy. Even so, ER-phagy requires neither the core autophagy machinery nor several other proteins specifically implicated in microautophagy. Thus, autophagy of ER whorls represents a distinct type of organelle-selective autophagy. Finally, we provide evidence that ER-phagy degrades excess ER membrane, suggesting that it contributes to cell homeostasis by controlling organelle size.

Keywords: Autophagy; Endoplasmic reticulum; Stress response.

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Figures

Fig. 1.
Fig. 1.
ER stress induces autophagy of ER whorls. (A) Electron micrograph of untreated wild-type yeast. Black arrows indicate cortical ER elements. (B) Wild-type yeast treated with DTT for 3 h. Note the expanded peripheral ER (black arrows), cytoplasmic ER extensions (white arrows) and ring-shaped ER whorls inside the vacuole. (C,D) ER whorls at the cell cortex. Micrographs are from cells treated with DTT for 1 h. (E,F) Engulfment of ER whorls by the vacuolar membrane. Micrographs are from cells treated with DTT for 4 h, but similar uptake intermediates were observed from 1 h onwards. In E, note the invagination of the vacuolar membrane during whorl uptake. In F, note the continuity between the peripheral ER and an ER whorl that is partially sequestered in a vacuolar membrane invagination (white arrows). (G,H) ER whorls in the vacuole. Micrographs are from cells treated with DTT for 3 h. CW, cell wall; Cy, cytoplasm; N, nucleus; V, vacuole.
Fig. 2.
Fig. 2.
ER stress activates both autophagy of ER whorls and non-selective macroautophagy. (A) Quantification of ER whorls in the cytoplasm, ER whorls engulfed by vacuolar membrane and ER whorls inside the vacuole in wild-type yeast treated with DTT for up to 4 h. 50 whorls were counted per time point and the fraction of cytoplasmic, engulfed and vacuolar whorls was determined. (B,C) Electron micrographs of wild-type yeast treated with DTT for 4 h. In B, black arrows indicate autophagic bodies containing cytoplasm. C shows autophagic bodies (black arrows) and an ER whorl (white arrow) in the same vacuole. Cy, cytoplasm; N, nucleus; V, vacuole.
Fig. 3.
Fig. 3.
Autophagy of ER whorls does not require the core autophagy machinery. (A) Electron micrographs of ER whorls in the vacuoles of Δatg1, Δatg7, Δatg8 and Δatg16 cells treated with DTT for 3 h. (B) Electron micrographs of serial 70-nm thin sections showing an ER whorl that has been taken up into the vacuole of a Δatg8 cell treated with DTT for 3 h. (C) Quantification of ER whorls in the vacuole in cells treated with DTT for 3 h. 30 whorls were counted per strain and the fraction of whorls in the vacuole was determined. Whorls engulfed by the vacuolar membrane were counted as cytoplasmic whorls.
Fig. 4.
Fig. 4.
ER stress triggers a composite autophagic response. (A) CPY activity in enzyme units (U) in tunicamycin-treated and DTT-treated wild-type cells over time. (B) Western blot of GFP from wild-type (WT), Δatg7 and PMSF-treated Δpep4 cells expressing GFP–Atg8 and treated with tunicamycin for the times indicated. Tunicamycin induces the ATG8 promoter so that the overall GFP signal increases with time. (C) As in B, but with strains expressing Sec63–GFP. Asterisks indicate Sec63–GFP fragments of ∼76, 63 and 59 kDa, whose generation required vacuolar proteolysis but not the core autophagy machinery. The GFP-containing cytosolic domain of Sec63–GFP is 76 kDa. (D) As in B but with strains expressing Rtn1–GFP. Tm, tunicamycin.
Fig. 5.
Fig. 5.
ER stress-induced autophagy selectively targets the ER and does not require the core autophagy machinery. (A) Schematics of the ER-localized reporters, all of which expose Pho8Δ60 to the cytosol. (B) Fold change of Pho8 activity of reporters for autophagy of cytosol (cyto-Pho8Δ60), mitochondria (mito-Pho8Δ60) and ER (Sec63–Pho8Δ60, Sec66–Pho8Δ60, Rtn1–Pho8Δ60 and Yop1–Pho8Δ60) upon nitrogen starvation in wild-type (WT) cells (blue bars) and Δatg7 cells (dark red bars). Data are mean±s.e.m., n = 3. (C) Fold change of Pho8 activity of reporters for autophagy of cytosol (cyto-Pho8Δ60) and ER (Sec66–Pho8Δ60, Rtn1–Pho8Δ60 and Yop1–Pho8Δ60) upon nitrogen starvation in WT (blue bars) and PMSF-treated Δpep4 cells (orange bars). (D) Fold change of Pho8 activity of the same reporters as in B upon tunicamycin treatment. Data are mean±s.e.m., n = 5. (E) Fold change of Pho8 activity of the same reporters as in C upon tunicamycin treatment.
Fig. 6.
Fig. 6.
Autophagy of ER does not require known autophagic machinery. (A) Fold change of Pho8 activity of Yop1–Pho8Δ60 after tunicamycin treatment in wild-type (WT) cells (blue bar) and Δatg1, Δatg6, Δatg7, Δatg8, Δatg14 and Δatg16 cells (dark red bars). Data are mean±s.e.m., n = 3. (B) Fold change of Pho8 activity of Yop1–Pho8Δ60 after tunicamycin treatment in wild-type (WT) cells (blue bar) and Δatg7 cells with or without additional deletion of EGO1, EGO3, VTC4, NVJ1 or PEP4 (dark red bars). Data are mean±s.e.m., n = 5. (C) Electron micrographs of ER whorls in the vacuoles of Δvps4 and Δvps23 cells treated with DTT for 3 h.
Fig. 7.
Fig. 7.
Degradation of excess ER by selective autophagy. (A) Electron micrographs of untreated Δopi1 and Δopi1 Δpep4 Δprb1 cells. Cw, cell wall; Cy, cytoplasm; V, vacuole. (B) Confocal images of Δopi1 Δatg7 Δpep4 Δprb1 and Δatg7 Δpep4 Δprb1 cells expressing the ER marker ssGFP-HDEL. Cells were stained with the vacuolar membrane dye FM4-64 but otherwise untreated. Arrows point to ER inside the vacuole where it colocalizes with vacuolar membrane. Scale bar: 2 µm.
Fig. 8.
Fig. 8.
Model of the autophagic response to ER stress. ER stress induces expansion of the peripheral ER (blue) and formation of ER whorls, which are selectively taken up into the vacuole by ER-phagy. Concomitantly, macroautophagy is activated. Forming autophagosomes (crescent-shaped membrane sac) engulf pieces of the ER (blue) as well as other cytoplasmic constituents (gray). ER-phagy and macroautophagy might act independently and in parallel, as shown in this model, but could also be linked.
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