COPII vesicles contribute to autophagosomal membranes

Abstract

A hallmark of autophagy is the de novo formation of double-membrane vesicles called autophagosomes, which sequester various cellular constituents for degradation in lysosomes or vacuoles. The membrane dynamics underlying the biogenesis of autophagosomes, including the origin of the autophagosomal membrane, are still elusive. Although previous studies suggested that COPII vesicles are closely associated with autophagosome biogenesis, it remains unclear whether these vesicles serve as a source of the autophagosomal membrane. Using a recently developed COPII vesicle-labeling system in fluorescence and immunoelectron microscopy in the budding yeast Saccharomyces cerevisiae, we show that the transmembrane cargo Axl2 is loaded into COPII vesicles in the ER. Axl2 is then transferred to autophagosome intermediates, ultimately becoming part of autophagosomal membranes. This study provides a definitive answer to a long-standing, fundamental question regarding the mechanisms of autophagosome formation by implicating COPII vesicles as a membrane source for autophagosomes.

Figure 1.

The COPII transmembrane cargo Axl2 is transferred from the ER to Atg8-positive structures. (A) Schematic diagram of the method for intense labeling of COPII vesicles. (B) sec12^ts SSA1pro-AXL2-GFP cells were grown at 23°C, incubated at 37°C for 1 h, and then incubated at 23°C in the presence of rapamycin. After a 2-h incubation, the cells were observed under a fluorescence microscope. Scale bars, 5 µm. (C) sec12^ts SSA1pro-AXL2-GFP cells were incubated at 37°C for 1 h and then treated with rapamycin at 23°C for 30 min, followed by fluorescence microscopy. Arrowheads show mCherry-Atg8 puncta positive for Axl2-GFP. Scale bars, 5 µm. The graph shows the proportion of mCherry-Atg8 puncta that were positive for Axl2-GFP. Values are means ± SD (n = 3). ***, P < 0.001 (unpaired two-tailed t test).

Figure 2.

COPII vesicles are recruited to forming autophagosomes. (A) Axl2 localization to Atg8 puncta in atg2Δ cells was examined as described in Fig. 1 C. Scale bars, 5 µm. Values in the graph are means ± SD (n = 3). ***, P < 0.001 (unpaired two-tailed t test). (B) sec12^ts SSA1pro-AXL2-GFP cells overexpressing Ape1 were incubated at 37°C for 1 h, transferred to nitrogen-starvation medium at 23°C for 1 h, and then observed by fluorescence microscopy. Arrowheads show an Axl2-GFP punctum on an isolation membrane visualized with mCherry-Atg8. Scale bars, 5 µm. The graph shows the proportion of Atg8-positive isolation membranes that colocalized with Axl2-GFP. Values are means ± SD (n = 3). ***, P < 0.001 (unpaired two-tailed t test). (C–E) sec12^ts SSA1pro-AXL2-GFP ypt7Δ cells were incubated at 37°C for 1 h, transferred to nitrogen-starvation medium at 23°C for 2 h, and then observed by fluorescence microscopy. Arrowheads show Axl2-positive Atg8 puncta. Scale bars, 5 µm. The graphs show the proportion of mCherry-Atg8 puncta that colocalized with Axl2-GFP (D) and the number of Atg8 puncta per cell (E). Values represent means ± SD (n = 3). ***, P < 0.001 (unpaired two-tailed t test).

Figure 3.

COPII vesicles become part of autophagosomal membranes. (A–J) sec12^ts SSA1pro-AXL2-GFP ypt7Δ cells were incubated at 37°C for 1 h, and then autophagy was induced at 23°C by nitrogen starvation for 3 h, followed by immunoelectron microscopy using anti-GFP antibodies. Open arrowheads indicate autophagosomes, and closed arrowheads indicate GFP signals (gold particles). Scale bars: 500 nm (A), 200 nm (B–J). (K) At least 30 autophagosomes in ERV14 and erv14Δ cells were examined, and the proportion of autophagosomes that contained gold particles on their membranes is shown in the graph.