Plant Cell Reports (2018) 37:653–664
Autophagy-related (ATG) 11, ATG9 and the phosphatidylinositol
3-kinase control ATG2-mediated formation of autophagosomes
· Kwang Deok Shin
· Jeong Hun Kim
· Taijoon Chung
Received: 28 November 2017 / Accepted: 11 January 2018 / Published online: 19 January 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Key message Using quantitative assays for autophagy, we analyzed 4 classes of atg mutants, discovered new atg2
phenotypes and ATG gene interactions, and proposed a model of autophagosome formation in plants.
Abstract Plant and other eukaryotic cells use autophagy to target cytoplasmic constituents for degradation in the vacuole.
Autophagy is regulated and executed by a conserved set of proteins called autophagy-related (ATG). In Arabidopsis, sev-
eral groups of ATG proteins have been characterized using genetic approaches. However, the genetic interactions between
ATG genes have not been established and the relationship between diﬀerent ATG groups in plants remains unclear. Here
we analyzed atg2, atg7, atg9, and atg11 mutants and their double mutants at the physiological, biochemical, and subcel-
lular levels. Involvement of phosphatidylinositol 3-kinase (PI3K) in autophagy was also tested using wortmannin, a PI3K
inhibitor. Our mutant analysis using autophagy markers showed that atg7 and atg2 phenotypes are more severe than those
of atg11 and atg9. Unlike other mutants, atg2 cells accumulated several autophagic vesicles that could not be delivered to
the vacuole. Analysis of atg double mutants, combined with wortmannin treatment, indicated that ATG11, PI3K, and ATG9
act upstream of ATG2. Our data support a model in which plant ATG1 and PI3K complexes play a role in the initiation of
autophagy, whereas ATG2 is involved in a later step during the biogenesis of autophagic vesicles.
Keywords Autophagosome · Phagophore · GFP-ATG8 · Phosphatidylethanolamine · atg18a
Cells possess enzymes to break down a variety of macro-
molecules. These enzymes are important for nutrient recy-
cling and the elimination of toxic and damaged molecules.
To prevent nonspeciﬁc degradation, catabolic activities in
eukaryotic cells are tightly regulated and isolated either in
specialized protein complexes (such as the proteasome) or in
membrane-bound lytic compartments (the vacuole in plants
and yeasts or the lysosome in metazoans).
Autophagy is a membrane traﬃcking route by which
cytoplasmic materials are delivered to the vacuole/lysosome
for degradation (Carlsson and Simonsen 2015; Michaeli
et al. 2016). Autophagy is initiated at the phagophore, a
membrane cisterna that subsequently expands to sequester
a portion of the cytoplasm. The sequestering activity of the
phagophore is completed when the growing border closes
via membrane scission to generate a double-membrane
structure called the autophagosome. After the autophago-
some matures, its outer membrane fuses with the vacuolar
membrane to release the autophagic body into the vacuolar
lumen. Finally, the autophagic body is rapidly degraded by
acid hydrolases in the vacuole. To observe autophagic bod-
ies in plant cells, the low pH of the vacuole needs to be
increased by treatment with concanamycin A (ConA), an
inhibitor of vacuolar proton pumps.
Communicated by Youn-Il Park.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00299-018-2258-9) contains
supplementary material, which is available to authorized users.
* Taijoon Chung
Department of Biological Sciences, Pusan National
University, Geumjeong-gu, Busan 46241, Republic of Korea
Institute of Systems Biology, Pusan National University,
Geumjeong-gu, Busan 46241, Republic of Korea