B. Buerte , Zhanghui Zeng, Chun Zhou, Guiwei Lian, Fu Guo, Junhui Wang, Ning Han, Muyuan Zhu, Hongwu Bian, Identification of New ATG8s-binding Proteins with Canonical LC3-interacting Region in Autophagosomes of Barley Callus, Plant and Cell Physiology, Volume 63, Issue 4, April 2022, Pages 508–520, https://doi.org/10.1093/pcp/pcac015
Plant and Cell Physiology
Published: 03 February 2022
Abstract
Autophagy is essential to maintain cellular homeostasis for normal cell growth and development. In selective autophagy, ATG8 plays a crucial role in cargo target recognition by binding to various adaptors and receptors with the ATG8-interacting motif, also known as the LC3-interacting region (LIR). However, the process of autophagy in the callus, as a proliferating cell type, is largely unknown. In this study, we overexpressed green fluorescent protein (GFP)-ATG8a and GFP-ATG8b transgenic barley callus and checked their autophagic activities. We identified five new ATG8 candidate interactors containing the canonical LIR motif by using immunoprecipitation coupled with mass spectrometry: RPP3, COPE, NCLN, RAE1, and CTSL. The binding activities between these candidate interactors and ATG8 were further demonstrated in the punctate structure. Notably, RPP3 was colocalized in ATG8-labeled autophagosomes under tunicamycin-induced ER stress. GST pull-down assays showed that the interaction between RPP3 and ATG8 could be prevented by mutating the LIRs region of RPP3 or the LIR docking site (LDS) of ATG8, suggesting that RPP3 directly interacted with ATG8 in an LIR-dependent manner via the LDS. Our findings would provide the basis for further investigations on novel receptors and functions of autophagy in plants, especially in the physiological state of cell de-differentiation.
Resistant callus selection and GFP imaging
After 8–10 weeks of culture on hygromycin-B selection medium, GFP fluorescence of hygromycin-resistant callus of GFP-ATG8a and GFP-ATG8b were imaged using a LUYOR-3415RG hand-held GFP exciter lamp (LUYOR, Shanghai, China). Photographs were taken using a 5D Mark III camera equipped with a LUV-495 filter from LUYOR.
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