Xiaohong Zhuang, Kin Pan Chung, Yong Cui, Weili Lin, Caiji Gao, Byung-Ho Kang, and Liwen Jiang

Significance

One fundamental question in the autophagy field is the membrane origin of the autophagosome. As the sole transmembrane autophagy-related (ATG) protein, ATG9 is conserved among eukaryotes and known to be important for autophagy, but its precise molecular function is still unknown. Through a combination of in vivo real-time imaging, 3D tomographic reconstruction, and genetic approaches, this study demonstrates that, in contrast to the atg9 mutants characterized in yeast and animal, loss of ATG9 in Arabidopsis led to expanding autophagosome-related tubules connected to the endoplasmic reticulum during autophagy. This work thus provides functional evidence for a unique role of ATG9 in autophagosome progression from the endoplasmic reticulum in plant cells, shedding new light on the membrane origins of autophagosome in plants.

Abstract

Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate–dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.

See: http://www.pnas.org/content/114/3/E426.full

PNAS January 17 2017; vol.114; no.3: E426–E435

Fig. 1.

Dysfunction of ATG9 leads to accumulation of autophagosome-related tubular structures upon BTH induction. (A) YFP-ATG8e–labeled tubules accumulate in atg9-3 after BTH treatment. Four-day-old YFP-ATG8e or YFP-ATG8e/atg9-3 seedlings were exposed to medium without BTH (Top) or with BTH (Middle) for 6 h and visualized under the confocal microscope. The YFP-ATG8e–positive dots are indicated by arrowheads whereas ring-like structures are indicated by arrows and enlarged in the Insets. (Bottom) FM4-64 was applied to label the tonoplast for 1 h, followed with additional BTH and Conc A treatment for 6 h before observation. v, vacuole. The number of autophagosome-related punctae or abnormal tubular structures per root section by Z stack projection with/without BTH treatment is quantified on the Right. The results were obtained from more than 10 individual seedlings (error bars ± SD). (Scale bars: 10 μm.) (B) Wortmannin treatment blocks the formation of YFP-ATG8e tubular structures in atg9-3 mutant. Four-day-old YFP-ATG8e/atg9-3 seedlings were transferred to medium with or without BTH for 6 h, respectively. Additional wortmannin was applied for 2 h after 4-h BTH treatment for subsequent confocal imaging. Ten slices were collected in a total thickness of 5.46 μm for generating the 3D projection image. (Scale bars: 10 μm.) Consistent results were obtained from at least three independent experiments. (C) Immunoblot detection of the vacuolar delivery of YFP core in YFP-ATG8e and YFP-ATG8e/atg9-3 before/after BTH induction. Total proteins were subjected to immunoblot analysis with GFP antibodies. Immunoblotting with cFBPase antibodies was used as a loading control. h, hour. Consistent results were obtained from three independent experiments. (D) Immunoblot detection of the ATG8 lipidation level in WT, atg9-3, and atg5-1. WT, atg9-3, and atg5-1 seedlings were incubated in medium with/without BTH and Conc A treatment for 6 h, respectively. Membrane fractions were subjected to immunoblot analysis with ATG8 antibodies. Immunoblotting with cFBPase antibodies was used as a loading control. Consistent results were obtained from three independent experiments.